Modified Monocytes/Macrophage Expressing Chimeric Antigen Receptors and Uses Thereof

ABSTRACT

The present invention includes methods and compositions for treating cancer, whether a solid tumor or a hematologic malignancy. By expressing a chimeric antigen receptor in a monocyte, macrophage or dendritic cell, the modified cell is recruited to the tumor microenvironment where it acts as a potent immune effector by infiltrating the tumor and killing the target cells. One aspect includes a modified cell and pharmaceutical compositions comprising the modified cell for adoptive cell therapy and treating a disease or condition associated with immunosuppression.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a 35 U.S.C. § 371 national phase applicationfrom, and claims priority to, International Application No.PCT/US2016/044440, filed Jul. 28, 2016, and published under PCT Article21(2) in English, which is entitled to priority under 35 U.S.C. § 119(e)to U.S. Provisional Application No. 62/197,675, filed Jul. 28, 2015, allof which are incorporated herein by reference in their entireties.

BACKGROUND OF THE INVENTION

Cancer immunotherapy has demonstrated exciting clinical results in thesetting of numerous solid tumors and hematologic malignancies. Theendogenous immune system is typically non-reactive to malignant cells,or can be actively immunosuppressive with respect to the body's reactionto the presence of malignant cells. One way to enhance treatment oftumors is to force tumor recognition by the immune system throughgenetic engineering of leukocytes. T cells can be engineered to expressa synthetic immunoreceptor comprising an extracellular targeted antibodyand intracellular signaling domain, known as chimeric antigen receptor(CAR). T cells expressing a CAR directed against CD19 have been shown tohave profound antileukemic efficacy, where complete remission has beenachieved in 90% of acute lymphoblastic leukemia patients treated (Maude,et al., NEJM, vol. 371:1507-17, 2014). These results are accompanied byrobust T cell proliferation and clearly documented T cell infiltrationinto tumor sites in leukemic patients so treated. Despite the highresponse rates demonstrated in hematopoietic malignancies, CAR T cellefficacy in solid tumors (as well as in certain lymphoid tumors) may belimited. Possible explanations for this include the potentially impairedability of T cells to infiltrate solid tumors, poor trafficking,immunosuppressive tumor microenvironment, and expression of few tumorspecific antigenson solid tumor cells.

A need exists in the art for more effective compositions and methodsthat treat cancers by improving specificity for tumor cells andimproving infiltration into tumor sites in both solid tumors andhematologic malignancies by such compositions. The present inventionfulfils this need.

SUMMARY OF THE INVENTION

As disclosed herein, the present invention includes compositions andmethods of using a phagocytic cell with targeted effector activity.

In one aspect, the invention includes a modified cell comprising achimeric antigen receptor (CAR), wherein the CAR comprises an antigenbinding domain, a transmembrane domain and an intracellular domain of astimulatory and/or co-stimulatory molecule, and wherein cell is amonocyte, macrophage, or dendritic cell that possesses targeted effectoractivity.

In another aspect, the invention includes a modified cell comprising anucleic acid sequence encoding a chimeric antigen receptor (CAR),wherein nucleic acid sequence comprises a nucleic acid sequence encodingan antigen binding domain, a nucleic acid sequence encoding atransmembrane domain and a nucleic acid sequence encoding anintracellular domain of a stimulatory and/or co-stimulatory molecule,and wherein the cell is a monocyte, macrophage, or dendritic cell thatexpresses the CAR and possesses targeted effector activity.

In yet another aspect, the invention includes a method of modifying acell comprising introducing a chimeric antigen receptor (CAR) into themonocyte, macrophage, or dendritic cell, wherein the CAR comprises anantigen binding domain, a transmembrane domain and an intracellulardomain of a stimulatory and/or co-stimulatory molecule, and wherein thecell is a monocyte, macrophage, or dendritic cell that expresses the CARand possesses targeted effector activity.

In still another aspect, the invention includes a composition comprisingthe cell modified according the method described herein.

In various embodiments of the above aspects or any other aspect of theinvention delineated herein, the antigen binding domain of the CARcomprises an antibody selected from the group consisting of a monoclonalantibody, a polyclonal antibody, a synthetic antibody, human antibody,humanized antibody, single domain antibody, single chain variablefragment, and antigen-binding fragments thereof. In another embodiment,the antigen binding domain of the CAR is selected from the groupconsisting of an anti-CD19 antibody, an anti-HER2 antibody, and afragment thereof. In yet another embodiment, the intracellular domain ofthe CAR comprises dual signaling domains.

In another embodiment, the targeted effector activity is directedagainst an antigen on a target cell that specifically binds the antigenbinding domain of the CAR. In yet another embodiment, the targetedeffector activity is selected from the group consisting of phagocytosis,targeted cellular cytotoxicity, antigen presentation, and cytokinesecretion.

In another embodiment, the composition further comprises an agentselected from the group consisting of a nucleic acid, an antibiotic, ananti-inflammatory agent, an antibody or antibody fragments thereof, agrowth factor, a cytokine, an enzyme, a protein, a peptide, a fusionprotein, a synthetic molecule, an organic molecule, a carbohydrate orthe like, a lipid, a hormone, a microsome, a derivative or a variationthereof, and any combination thereof.

In another embodiment, the modified cell has at least one upregulated M1marker and at least one downregulated M2 marker. In yet anotherembodiment, the modified cell is genetically modified to express theCAR. In still another embodiment, the targeted effector activity isenhanced by inhibition of CD47 or SIRPa activity.

In another embodiment, introducing the CAR into the cell comprisesintroducing a nucleic acid sequence encoding the CAR, such aselectroporating a mRNA encoding the CAR or transducing the cell with aviral vector comprising the nucleic acid sequence encoding the CAR.

In another embodiment, the targeted effector activity is directedagainst an antigen on a target cell that specifically binds the antigenbinding domain of the CAR. In another embodiment, the targeted effectoractivity is selected from the group consisting of phagocytosis, targetedcellular cytotoxicity, antigen presentation, and cytokine secretion.

In another embodiment, the method described herein further comprisesinhibiting CD47 or SIRPα activity to enhance the targeted effectoractivity, such as by contacting the cell with a blocking anti-CD47 or ablocking anti-SIRPα antibody. In yet another embodiment, the methodfurther comprises modifying the cell to deliver an agent to a target,wherein the agent is selected from the group consisting of a nucleicacid, an antibiotic, an anti-inflammatory agent, an antibody or antibodyfragments thereof, a growth factor, a cytokine, an enzyme, a protein, apeptide, a fusion protein, a synthetic molecule, an organic molecule, acarbohydrate or the like, a lipid, a hormone, a microsome, a derivativeor a variation thereof, and any combination thereof.

In one aspect, the invention includes a pharmaceutical compositioncomprising the cell described herein.

In another aspect, the invention includes a use of the modified celldescribed herein in the manufacture of a medicament for the treatment ofan immune response in a subject in need thereof. In yet another aspect,the invention includes a use of the modified cell described herein inthe manufacture of a medicament for the treatment of a tumor or cancerin a subject in need thereof.

In yet another aspect, the invention includes a method of treating adisease or condition associated with a tumor or cancer in a subjectcomprising administering to the subject a therapeutically effectiveamount of a pharmaceutical composition comprising the modified celldescribed herein.

In still another aspect, the invention includes a method of treating atumor in a subject, comprising administering to the subject atherapeutically effective amount of a pharmaceutical compositioncomprising the modified cell described herein.

In another aspect, the invention includes a method for stimulating animmune response to a target tumor cell or tumor tissue in a subjectcomprising administering to a subject a therapeutically effective amountof a pharmaceutical composition comprising the modified cell describedherein.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of preferred embodiments of theinvention will be better understood when read in conjunction with theappended drawings. For the purpose of illustrating the invention, thereare shown in the drawings embodiments which are presently preferred. Itshould be understood, however, that the invention is not limited to theprecise arrangements and instrumentalities of the embodiments shown inthe drawings.

FIG. 1A is a series of images showing the conceptual diagram of achimeric antigen receptor (CAR) comprised of a gene/gene-productcontaining an extracellular domain with targeting function, a hingedomain, a transmembrane domain, an intracellular signaling domain(s),and/or a 2A (P2A, T2A) for stoichiometric co-expression of an additionalgene product which may or may not be secreted, including anygene/transcript/protein, including but not limited to a cytokine,monoclonal antibody, antibody fragment, single chain variable fragment,enzyme, additional receptor, dominant negative receptor, tumorassociated antigen(s), and any combination thereof. In addition, the CARconstruct may include co-delivery of CRISPR/Cas9 gene editing material,or be introduced in the context of a CRISPR/Cas9 pre-edited cell.

FIG. 1B is a series of images showing specific examples of CARconstructs, including CARMA-ζ, CARMA-γ, and CARMA-Dectin, which containan antigen specific scFv, CD8 hinge, CD8 transmembrane, and a CD3 ζ,FcεRI common γ subunit, or the intracellular domain of Dectin-1,respectively.

FIG. 2A is a graph showing CAR19z expressed on the surface of myeloidcells post lentiviral transduction. CAR19z lentivirus was titrated inthree-fold dilutors and used to transduce 1e5/0.1 mL mRFP+THP1 cells.mRFP is a reporter gene (red fluorescent protein) that was expressed bylentiviral transduction of the myeloid cell line THP1. These cells canbe induced to differentiate to macrophages upon exposure to the chemicalPMA. THP1 cells were harvested 24 hours post-transduction and stainedfor CAR surface expression with biotinylated-protein L followed bystreptavidin-APC.

FIG. 2B is a graph showing transduced THP1 cells expanded and sorted byFACS to generate a 100% CAR19z positive mRFP+THP1 subline.

FIG. 2C demonstrates expression of anti-CD19, anti-HER2, andanti-mesothelin lentiviral CAR constructs on THP1 macrophages, withCAR(+) events in the upper right quadrant.

FIG. 3A is a flow chart showing the overview of CARMA subline generationusing a THP1 macrophage model, differentiation with 1 ng/mL phorbol12-myristate 13-acetate (PMA), and in vitro phagocytosis assay.

FIG. 3B is a graph showing anti-CD19 CAR macrophages, but not wild type(Wt) macrophages, phagocytosed K562 tumor cells that expressed CD19, asdemonstrated by fluorescent microscopy based phagocytosis assays.

FIG. 3C is a graph showing anti-HER2 CAR macrophages, but not wild type(Wt) macrophages, phagocytosed K562 tumor cells that expressed HER2, asdemonstrated by fluorescent microscopy based phagocytosis assays.

FIG. 3D is a graph showing anti-mesothelin CAR macrophages, but not wildtype (Wt) macrophages, phagocytosed K562 tumor cells that expressedmesothelin, as demonstrated by fluorescent microscopy based phagocytosisassays.

FIG. 3E is a representative FACS plot showing CARMA tumor phagocytosiswas validated by a flow cytometric based assay, in which mRFP+CARMAagainst CD19 were co-cultured with CD19+ GFP+K562 cells and doublepositive events were quantified.

FIG. 3F is an image showing mRFP in a standard 10× field of view used inthe tabulation of CARMA phagocytosis function.

FIG. 3G is an image showing an overlay in a standard 10× field of viewused in the tabulation of CARMA phagocytosis function.

FIG. 3H is a series of images showing FACS based mRFP/GFP doublepositive events were defined as phagocytic events, and were validated assuch by Amnis Imagestream FACS analysis. Events shown are gated ondouble positive events and ordered from high to low by the AmnisImagestream phagocytosis-erode algorithm.

FIG. 3I is a series of images showing phagocytosis of tumor cells bymRFP+CARMA in the THP-1 cell line model was further demonstrated byconfocal microscopy, verifying that GFP+ tumor cells have beencompletely enclosed within phagosomes via three-dimensional confocalz-stack reconstructions.

FIG. 3J is a series of images showing phagocytosis of tumor cells bymRFP+CARMA in the THP-1 cell line model was further demonstrated byconfocal microscopy, verifying that GFP+ tumor cells have beencompletely enclosed within phagosomes via three-dimensional confocalz-stack reconstructions.

FIG. 3K is a series of images that demonstrate the fate of a singleCARMA cell over time—with contact and immunological synapse formationbeing the first step, leading to phagocytic engulfment, degradation oftumor using loss of GFP as a marker of cell death, phagosome breakdown,and phagosome repair—demonstrating that CARMA survive post tumor cellphagocytosis.

FIG. 4A is a graph showing anti-CD19 CAR macrophages tested using in anin vitro phagocytosis assay against CD19+(target) or CD19−(control)GFP+K562 tumor cells. Demonstrating the antigen specificity of CARMA,only antigen-bearing tumor cells were phagocytosed. To demonstrate therequirement for the intracellular signaling domain in CARMA function,CAR19-Δζ constructs (which lack an intracellular signaling domain) wereutilized.

FIG. 4B is a graph showing CAR19-Δζ macrophages failed to phagocytosetumor cells.

FIG. 4C is a graph showing the CAR19-Δζ macrophages had significantlyreduced anti-tumor function via an in vitro luciferase based specificlysis assay.

FIG. 4D is a graph showing in vitro CARMA phagocytosis assay performedin the presence of R406 (Syk inhibitor). R406 independently abrogatedthe phagocytic function of CARMA, indicating that CAR signaling inmacrophages is Syk dependent and results in actin polymerization andNMIIA mediated phagocytic function.

FIG. 4E is a graph showing in vitro CARMA phagocytosis assay performedin the presence of cytochalasin D (actin polymerization inhibitor).Cytochalasin D independently abrogated the phagocytic function of CARMA,indicating that CAR signaling in macrophages is Syk dependent andresults in actin polymerization and NMIIA mediated phagocytic function.

FIG. 4F is a graph showing in vitro CARMA phagocytosis assay performedin the presence blebbistatin (non-muscle myosin IIA inhibitor).Blebbistatin independently abrogated the phagocytic function of CARMA,indicating that CAR signaling in macrophages is Syk dependent andresults in actin polymerization and NMIIA mediated phagocytic function.

FIG. 5A is a flow cytometric graph showing expression of CD47 on targettumor cell lines relative to isotype control. K562 and K562-CD19+(K19)were used in these experiments, both of which are high CD47 expressingcell lines.

FIG. 5B is a graph showing that the addition of anti-CD47 monoclonalantibody selectively enhanced CAR but not Wt macrophage mediatedphagocytosis of target antigen bearing tumor cells. Wt or CAR19ζmacrophages were incubated with CD19+K562 tumor cells either with 0,0.01, 0.10, 1.00, or 10.0 mcg/mL anti-CD47 monoclonal antibody.

FIG. 5C is a graph showing that the addition of anti-SIRPα monoclonalantibody selectively enhanced CAR but not Wt macrophage mediatedphagocytosis of target antigen bearing tumor cells. Wt or CAR19ζmacrophages were incubated with CD19+K562 tumor cells either with 0,0.01, 0.10, 1.00, or 10.0 mcg/mL anti-SIRPα monoclonal antibody.

FIG. 5D is a graph demonstrating that blockade of the CD47/SIRPα axiswith anti-SIRPα monoclonal antibodies enhanced the polyphagocytic(defined as a macrophage that has engulfed 2 or more tumor cells atonce) by CAR macrophages.

FIG. 5E is a graph showing an in vitro phagocytosis assay. To controlfor the added opsonization by the CD47/SIRPα blocking monoclonalantibodies, a control anti-CD47 monoclonal antibody (clone 2D3), whichbinds CD47 but does not block the CD47 to SIRPα binding site, was usedin an in vitro phagocytosis assay. Only the clone which blocked thebinding site (anti-CD47, clone B6H12) or blockade of the SIRPα receptordirectly lead to enhancement of CARMA tumor phagocytosis.

FIG. 5F is a graph showing an in vitro phagocytosis againstantigen-negative (CD19 negative) tumor cells. To test whether blockadeof the CD47/SIRPα axis on CAR macrophages leads to loss of antigenspecificity, an in vitro phagocytosis against antigen-negative (CD19negative) tumor cells was conducted in the presence of anti-CD47 oranti-SIRPα monoclonal antibody, and there was no observablephagocytosis.

FIG. 5G is a graph showing the specificity of CARMA phagocyticenhancement in the presence of SIRPα blocking monoclonal antibody testedby knocking out the SIRPα receptor on THP1 macrophages, and comparingtumor phagocytosis by CARMA or SIRPα-KO CARMA in the absence or presenceof anti-SIRPα antibody. CRISPR/Cas9 was used for SIRPα deletion, andcells were sorted for SIRPα negativity prior to functional assays.Knocking out SIRPα enhanced CARMA function, and adding anti-SIRPα backto the knockout cells failed to further enhance phagocytosis.

FIG. 6A is a graph showing the specific lysis of CD19+ GFP+Luciferase+K562 cells by CAR19ζ CARMA but not Wt macrophages (using theTHP-1 macrophage model) in an in vitro luciferase based killing assay at48 hours in a dose dependent manner.

FIG. 6B is a graph demonstrating the specific lysis of tumor cells byCAR19ζ or Wt THP-1 monocytes (undifferentiated, thus a model ofmonocytes rather than macrophages) in an in vitro luciferase basedkilling assay at 48 hours in a dose dependent manner.

FIG. 6C is a panel of images showing the luciferase drivenbioluminescence, derived from luciferase positive CD19+K562 tumor cells,after 48-hour co-culture with Wt or CAR19ζ macrophages in vitro, in theabsence or presence of 10 μg/mL anti-SIRPα monoclonal antibody.

FIG. 6D is a graph demonstrating the specific lysis of Wt or CAR19ζmacrophages +/−anti-SIRPα monoclonal antibody.

FIG. 7A is a series of graphs showing CAR constructs with an FcεRIcommon γ (CAR19γ, CARMA19γ) subunit intracellular domain were generated,packaged into lentivirus, and used to transduce THP-1 myeloid cells in athree-fold serial viral dilution. CAR19γ was expressed on THP-1macrophages.

FIG. 7B is a graph showing CAR19γ macrophages or CAR19ζ macrophagessorted for 100% CAR positivity and utilized for in vitro functionalcharacterization. CAR19ζ and CAR19γ macrophages both phagocytosed CD19+tumor cells, and both displayed synergy with blockade of the CD47/SIRPαaxis by the addition of anti-SIRPα monoclonal antibody

FIG. 7C is a graph showing an R406 Syk inhibition in vitro phagocytosisassay that demonstrates that CAR19ζ and CAR19γ macrophages both signalvia Syk to drive tumor phagocytosis.

FIG. 7D is a graph showing that both CAR19ζ and CAR19γ THP1 macrophages,but not Wt THP1 macrophages, efficiently killed CD19+ tumor cells in anin vitro luciferase-based specific lysis assay after 24 hours ofco-culture at various E:T ratios.

FIG. 8A is a graph showing macrophages responded to conserved molecularcues of infection, such as pathogen associated molecular patterns, viaconstitutively expressed pathogen recognition receptors.

FIG. 8B is a graph showing an in vitro phagocytosis assay conductedusing CAR macrophages that were primed with the ligands for TLR1-9,independently, or media control to enhance the tumor phagocytic functionof CARMA. Ligands for TLR1, 2, 4, 5, and 6 enhanced the phagocyticfunction of CARMA.

FIG. 8C is a graph showing the difference between TLR ligands thatenhanced or did not enhance CARMA phagocytosis of tumor cells in a rangeof TLR3 or TLR6 ligand concentrations.

FIG. 9A is a graph showing β-glucan, a yeast product, bound to Dectin-1on the surface of macrophages and resulted in activation and effectorfunction. In order to test the capacity of β-glucan to augment CARMAfunction, in vitro tumor phagocytosis assays were conducted in theabsence of presence of 5 mcg/mL β-glucan. β-glucan enhanced thephagocytic capacity of CAR, but not Wt, macrophages.

FIG. 9B is a series of graphs showing in vitro luciferase based specificlysis assays conducted at various effector (E):target (T) ratios in thepresence of 0, 0.5, 5, of 50 μg/mL β-glucan to test the capacity ofβ-glucan to enhance CARMA tumor killing. β-glucan enhanced the specificlysis of antigen bearing tumor cells by CAR but not Wt THP-1macrophages.

FIG. 10A is a series of images showing CAR constructs comprised of aDectin-1 intracellular signaling domain were generated. These constructswere packaged into lentivirus and used to transduce THP-1 myeloid cellsin a three-fold serial dilution of lentiviral titers.

FIG. 10B is a graph showing that CAR was detected on the surface ofmacrophage expressing CD8TM-Dectin1 CAR constructs.

FIG. 10C is a graph showing that CAR was detected on the surface ofmacrophage expressing DectinTM-Dectin1 CAR constructs.

FIG. 10D is a graph showing CD8TM-Dectin1 CAR and DectinTM-Dectin1 CARmacrophages were tested in an in vitro luciferase killing assay. Bothconstructs demonstrated specific lysis of tumor cells.

FIG. 10E is a graph showing Dectin1-CAR macrophages tested in an invitro tumor phagocytosis assay against K562 (control) or K19 (target)tumor cells. Dectin1-CAR macrophages selectively phagocytosedcognate-antigen bearing tumor cells.

FIG. 10F is a series of images showing Dectin-1 CAR macrophagesdemonstrated the capacity for phagocytosis of multiple tumor cells.

FIG. 10G is a graph showing an in vitro tumor phagocytosis assay.Dectin1-CAR macrophages demonstrated synergy with blockade of SIRPα, orwith priming with a TLR ligand.

FIG. 11A is a series of graphs showing calreticulin levels in threedifferent CD19+ target cell lines relative to isotype control.

FIG. 11B is a graph showing the normalized mean fluorescent intensity ofcalreticulin expression in three different CD19+ target cell lines.

FIG. 11C is a graph showing that low levels of calreticulin moderatelyprotected target cells, specifically Nalm6 and JEKO cell lines, fromCAR19z macrophage phagocytosis. These data suggest that exploitation ofcalreticulin deposition/induction can be used an additional tactic toaugment CARMA effector function.

FIG. 12A is a series of graphs showing anti-HER2 CAR constructs clonedinto mRNA expression plasmids, transcribed in vitro, and the mRNAdirectly electroporated into primary human monocytes.

FIG. 12B is a series of graphs showing the efficiency of anti-HER2 CARmRNA electroporation into primary human monocyte derived macrophages(fully differentiated) at 79.7%.

FIG. 12C is a graph showing that while mRNA electroporation results in ahigh CAR transfection efficiency of both monocytes and macrophages, CARexpression was temporary due to mRNA degradation, peaking at day 2 anddisappearing by day 7 post electroporation in vitro.

FIG. 13A is a graph showing NSGS mice injected with 1E6 SKOV3 CBG/GFP+human ovarian cancer cells via IP injection, a model of metastaticintraperitoneal carcinomatosis of HER2+ ovarian cancer. Mice wereco-injected with either mock electroporated or anti-HER2 CAR mRNAelectroporated primary human macrophages (1:1 E:T ratio) and tumorburden was imaged. CAR macrophages demonstrated marginal reduction intumor growth over approximately two weeks. The first time point at whichtumor burden was bioluminescently quantified was 24 hourspost-treatment, demonstrating that CAR monocytes and macrophages hadactivity in the first 24 hours.

FIG. 13B is a graph showing NSGS mice injected with 1E6 SKOV3 CBG/GFP+human ovarian cancer cells via IP injection, a model of metastaticintraperitoneal carcinomatosis of HER2+ ovarian cancer. Mice wereco-injected with either mock electroporated or anti-HER2 CAR mRNAelectroporated primary human monocytes (1:1 E:T ratio) and tumor burdenwas imaged. CAR monocytes demonstrated marginal reduction in tumorgrowth over approximately two weeks. The first time point at which tumorburden was bioluminescently quantified was 24 hours post-treatment,demonstrating that CAR monocytes and macrophages had activity in thefirst 24 hours.

FIG. 14A is a series of graphs showing lentiviral delivery of CARtransgenes to primary human monocyte derived macrophages was testedusing multiple CAR constructs. CAR19 was delivered to human macrophagesvia lentiviral transduction, demonstrating a 4.27% and 38.9%transduction efficiency in the control vs. CAR19 lentivirus (MOI 10)groups, respectively.

FIG. 14B is a series of representative FACS plots showing the expressionof anti-HER2 CAR in primary human macrophages, with a 1.47 and 18.1%transduction efficiency between the control and MOI 10 CAR LVconditions, respectively.

FIG. 15A is a series of graphs showing transduction efficiency peaked atthe midpoint of transduction (day 4), for anti-CD19. Monocyte derivedmacrophages were generated by differentiating CD14+ selected cells (fromnormal donor apheresis products) in GM-CSF conditioned media for 7 days.To optimize delivery of CAR via lentiviral transduction, anti-CD19lentivirus was used to transduce macrophages at different points of themonocyte to macrophage differentiation process.

FIG. 15B is a series of graphs showing transduction efficiency peaked atthe midpoint of transduction (day 4), for anti-HER2. Monocyte derivedmacrophages were generated by differentiating CD14+ selected cells (fromnormal donor apheresis products) in GM-CSF conditioned media for 7 days.To optimize delivery of CAR via lentiviral transduction, anti-HER2lentivirus was used to transduce macrophages at different points of themonocyte to macrophage differentiation process.

FIG. 15C is a series of graphs showing that the efficacy of phagocytosistrended with the CAR transduction efficiency, peaking with macrophagestransduced at day 4 during the differentiation process.

FIG. 16A is a series of graphs showing alternative transductionapproaches to delivering transgenes to primary human macrophages weretested, given that mRNA electroporation was transient and lentivirus wasonly moderately efficient and required high titer. Adenovirus(recombinant, replication deficient) was identified as an efficientapproach to primary human macrophage transduction. Expression ofCoxackie Adenovirus Receptor (the docking protein for Ad5) and CD46 (thedocking protein for Ad35) were tested relative to isotype control onprimary human macrophages, and CD46 but not Coxackie Adenovirus receptorwas highly expressed. Thus, chimeric Ad5f35 adenovirus was utilized forprimary human macrophage transduction, and was engineered via standardmolecular biology techniques to express a chimeric antigen receptor (GFPand empty Ad5f35 viruses were used as controls) against HER2.

FIG. 16B is a graph showing that at an MOI of 1000, Ad5f35 effectivelydelivered a transgene (GFP was used as a model transgene) into humanmacrophages, and expression went up over time as monitored by GFP signalquantification on an IVIS Spectrum.

FIG. 16C is a graph showing the comparison of the transduction kineticsof primary human macrophages at different timepoints across a broadrange of MOIs—up to 10,000.

FIG. 16D is a series of representative FACS plots of anti-HER2 CARexpression on Ad5f35 transduced human macrophages at 48 hours posttransduction, at a broad range of viral MOIs.

FIG. 16E is a series of representative fluorescent microscopy images ofAd5f35-GFP transduced primary human macrophages, with the highesttransduction efficiency demonstrated at an MOI of 1000.

FIG. 17A is a series of graphs showing primary human CARMA tested in anin vitro phagocytosis assay via FACS analysis. Macrophages (untransducedor anti-HER2 CAR) were stained with DiI prior to co-culture with GFP+SKOV3 ovarian cancer cells. Phagocytosis, defined by DiI/GFP doublepositive events, was measured at a level of 26.6% in the CAR group and4.55% in the control group.

FIG. 17B is a series of images demonstrating visually that these doublepositive events represent phagocytosis. To validate that the DiI/GFPdouble positive events were phagocytic events and not doublets,cytochalasin D (a phagocytosis inhibitor) was added to an arm of theexperiment, and fully abrogated CAR mediated phagocytosis down to 1.74%.To further validate that primary human CAR macrophages could phagocytosetumor cells, double positive events were gated by Amnis Imagestream FACSand ordered from high to low by the Amnis phagocytosis-erode algorithm.

FIG. 17C is a series of images showing confocal microscope images of DiIstained CAR-HER2 macrophages co-cultured with SKOV3-GFP.

FIG. 18 is a graph showing CAR, but not UTD, human macrophagesphagocytosed breast cancer cells. Anti-HER2 CAR primary humanmacrophages generated using Ad5f35-CAR transduction of monocyte derivedmacrophages. These cells (or control untransduced cells) were utilizedas effectors in an in vitro FACS based phagocytosis assay of SKBR3 humanbreast cancer cells. In addition, addition of anti-SIRPα monoclonalantibody enhanced CARMA but not UTD macrophage phagocytosis of breastcancer cells. These results demonstrate that the synergy betweenblockade of the CD47/SIRPα axis seen with CARMA in the THP-1 modeltranslates to primary human macrophage studies.

FIG. 19 is a series of representative FACS plot showing that CARMAexhibit intact phagocytosis of pH-Rodo Green E. Coli particles. In orderto demonstrate that CAR macrophages were still functional innate immunecells in the anti-microbial sense, and did not lose the capacity torespond to infectious stimuli, control untransduced or CAR macrophageswere employed in a FACS based E. Coli phagocytosis assay.

FIG. 20A is a graph showing primary human anti-HER2 CARMA tested aseffector cells in in vitro luciferase based killing assays. Anti-HER2CARMA, but not control UTD macrophages, led to the specific lysis ofHER2+K562 cells but not control K562 cells, lacking HER2 expression,after 48 hours of co-culture.

FIG. 20B is a graph showing in vitro luciferase based killing assayutilizing SKBR3 breast cancer cells as targets. CARMA, but not controlUTD or control Empty Ad5f35 transduced macrophages, had significantanti-tumor activity against both models after 48 hours of co-culture.

FIG. 20C is a graph showing in vitro luciferase based killing assayutilizing SKOV3 ovarian cancer cells as targets. CARMA, but not controlUTD or control Empty Ad5f35 transduced macrophages, had significantanti-tumor activity against both models after 48 hours of co-culture.

FIG. 20D is a graph showing the synergy between blockade of theCD47/SIRPα axis in a killing assay. SKOV3 ovarian cancer cells wereco-cultured with media, control untransduced macrophages, anti-HER2CARMA, anti-HER2 CARMA+antiCD47 mAB (10 mcg/mL), or anti-HER2CARMA+anti-SIRPα (10 mcg/mL) and luciferase signal was seriallymeasured. CARMA led to complete tumor eradication by day 13, while thekinetics of tumor eradication were even faster in the presence ofblocking the CD47/SIRPα axis.

FIG. 20E is a graph showing the synergy with β-glucan, which wasdemonstrated in a THP-1 macrophage CARMA model, and β-glucan priming ofthe CARMA led to enhanced tumor killing kinetics.

FIG. 20F is a graph showing that exposure of CARMA to LPS (a TLR-4ligand) or Poly-IC (a TLR-3 ligand) led to modulation of the anti-tumoreffect.

FIG. 21 is a series of images showing the capacity for primary humanCARMA to clear tumors in an in vitro luciferase assay. GFP+ SKOV3ovarian cancer cells were co-cultured with control UTD macrophages,control UTD macrophages plus 10 mcg/mL trastuzumab, control empty Ad5f35virus transduced macrophages, or anti-HER2 primary human CARMA. CARMA,but not the control conditions, were capable of clearing the tumorcells.

FIG. 22A is a panel of graphs showing a dose dependent up-regulation ofM1 markers CD80/CD86, and a dose dependent down-regulation of M2 markerCD163, were measured by FACS. Macrophages are phenotypically plasticcells capable of adopting diverse functional features, commonlyseparated into the M1 and M2 macrophage classifications—with M1 beinginflammatory/activated, and M2 being immunosuppressive/tumor-promoting.M1 and M2 markers were measured 48 hours after transduction of primaryhuman macrophages with Ad5f35 CAR virus.

FIG. 22B is a series of graphs showing whether the effect on M1 and M2markers was a result of CAR expression or Ad5f35 transduction.Macrophages were transduced with either nothing, empty Ad5f35, oranti-HER2 Ad5f35, and empty/CAR Ad5f35 showed the same pattern ofphenotype shift.

FIG. 22C is a graph showing that CARMA exposed to suppressive cytokinesmaintained their killing activity in a luciferase based in vitrospecific lysis assay at 48 hours. Control UTD macrophages wereconditioned with suppressive cytokines demonstrated enhanced tumorgrowth.

FIG. 22D is a panel of graphs showing the resistance toimmunosuppression of human CAR macrophages, control UTD, Empty Ad5f35,or anti-HER2 CAR Ad5f35 transduced macrophages exposed to 10 ng/mL ofIL-4, a canonical M2 inducing cytokine, or cancer cells that werepreviously shown to subvert macrophages to M2 during co-culture (SKOV3,ovarian cancer cell line; HDLM2, Hodgkin lymphoma cell line). ControlUTD macrophages upregulated CD206, an M2 marker that specificallyresponds to IL-4 stimulation via STATE phosphorylation. Empty Ad5f35,and more so CAR-Ad5f35 transduced macrophages, displayed resistance toIL-4 and tumor induced subversion to the M2 phenotype.

FIG. 22E is a graph showing metabolic phenotype of control UTD oranti-HER2 CAR macrophages exposed to IL-4 for 24 hours to polarize to M2(or not), and oxygen consumption rate.

FIG. 22F is a graph showing that phenotypic, metabolic, and functionalassays indicate that CARMA are resistant to M2 subversion.

FIG. 23A is a panel of graphs showing primary human normal donormonocytes (purified via CD14 positive selection) transduced withAd5f35-CAR-HER2 at MOI's ranging from 0 (UTD) to 1000. CAR expressionwas measured via FACS 48 hours post transduction. CAR monocytes wereefficiently generated with Ad5f35, with expression peaking at an MOI of1000.

FIG. 23B is a graph showing primary monocyte transduction efficiency.

FIG. 23C is a graph showing that monocytes maintained high viability(measured by FACS Live/Dead Aqua analysis) at MOIs up to 1000.

FIG. 23D is a series of graphs showing CAR but not untransduced (UTD)human monocytes upregulated M1 activation markers.

FIG. 23E is a series of graphs showing owing CAR but not untransduced(UTD) human monocytes downregulated M2 markers.

FIG. 24A is a graph showing anti-HER2 CAR monocyte killing of HER2+SKBR3 cells (human breast cancer) assessed via an in vitro luciferasebased killing assay.

FIG. 24B is a graph showing anti-HER2 CAR monocyte killing of HER2+SKOV3 cells (human ovarian cancer) assessed via an in vitro luciferasebased killing assay.

FIG. 25A is a schematic diagram of the NOD-scid IL2Rg-null-IL3/GM/SF,NSG-SGM3 (NSGS) mice used to model human HER2(+) ovarian cancerxenografts in vivo. On day 0 mice were injected intraperitoneally (IP)with 7.5E5 click beetle green luciferase (CBG luc) positive/greenfluorescent protein (GFP) positive SKOV3 ovarian cancer cells as a modelof intraperitoneal carcinomatosis, an aggressive inherently metastaticmodel of solid malignancy. Mice were either untreated (tumor alone), orinjected with a single dose of 4E6 untransduced (UTD) or CAR-HER2(CARMA) human macrophages on day 0 via IP injection.

FIG. 25B is a graph showing mice serially imaged using bioluminescence(total flux; photons/second) as a surrogate of tumor burden.

FIG. 25C is a graph showing percent survival of mice that received CARMAtreatment. CARMA treated mice had a decrease in tumor burden ofapproximately two orders of magnitude.

FIG. 25D is a panel of images showing that mice treated with CARMA had a30 day survival benefit (p=0.018) relative to untreated or UTDmacrophage treated mice.

FIG. 25E is a panel of graphs showing tumors harvested from mice thatdied on day 36 and assessed for the presence of adoptively transferredhuman macrophages via human CD45 expression on FACS analysis.

FIG. 26A is a graph showing surface CAR expression verified by FACSanalysis 48 hours post transduction of human macrophages eitheruntransduced (UTD) or transduced with empty Ad5f35 virions lacking atransgene (Empty) or Ad5f35-CAR-HER2-t (CARMA) at multiplicities ofinfection of 1000.

FIG. 26B is a panel of graphs showing surface markers assessed todemonstrate M1 macrophage polarization in cells transduced by eitherempty Ad5f35 or CAR-HER2-Ad5f35. M1 markers (HLA DR, CD86, CD80, PDL1)were upregulated while M2 markers (CD206, CD163) were downregulated

FIG. 26C is a schematic diagram of the NSGS mice used in an IP model ofHER2+ metastatic ovarian cancer, and stratified into four treatment arms(n=5 per arm). Mice were left untreated or given IP injections of 1E7untransduced, empty-Ad5f35 transduced macrophages, or CAR-HER2-ttransduced macrophages on day 0.

FIG. 26D is a panel of images showing tumor burden monitored via serialbioluminescent imaging, with representative data shown at day 27 posttumor engraftment.

FIG. 26E is a graph showing tumor burden monitored via serialbioluminescent imaging, with representative data shown at day 27 posttumor engraftment.

FIG. 27A is a schematic diagram of the NSGS mice used in an IP model ofHER2+ metastatic ovarian cancer, and stratified into four treatment arms(n=5 per arm), including no treatment, and either 3E6, 1E7, or 2E7CAR-HER2-ζ human macrophages, administered IP on day 0.

FIG. 27B is a graph showing tumor burden monitored via serialbioluminescent imaging. A dose-dependent response to the number ofmacrophage was observed in this model.

FIG. 27C is a graph showing that single doses of CAR-HER2 macrophages at3E6, 1E7, or 2E7 macrophages per mouse led to dose dependent tumoreradication (relative to untreated mice) by day 36 post engraftment.

FIG. 28 is an illustration of the proposed therapeutic approach forCARMA. In brief, patient monocytes would be selected from the peripheralblood, ex vivo differentiated and transduced to express a CAR,co-stimulated (or not) with synergistic compounds, and injected backinto the patient either intravenously, intraperitoneally,intratumorally, via interventional radiological procedure, or by otherroute. Of note, the differentiated process could be skipped andmonocytes can be transduced and infused back into the patient. Themonocyte source may also be an HLA matched donor.

DETAILED DESCRIPTION Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice for testing of the present invention, the preferredmaterials and methods are described herein. In describing and claimingthe present invention, the following terminology will be used.

It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

“About” as used herein when referring to a measurable value such as anamount, a temporal duration, and the like, is meant to encompassvariations of ±20% or ±10%, more preferably ±5%, even more preferably±1%, and still more preferably ±0.1% from the specified value, as suchvariations are appropriate to perform the disclosed methods.

“Activation,” as used herein, refers to the state of amonocyte/macrophage that has been sufficiently stimulated to inducedetectable cellular proliferation or has been stimulated to exert itseffector function. Activation can also be associated with inducedcytokine production, phagocytosis, cell signaling, target cell killing,or antigen processing and presentation. The term “activatedmonocytes/macrophages” refers to, among other things,monocyte/macrophage that are undergoing cell division or exertingeffector function.

The term “agent,” or “biological agent” or “therapeutic agent” as usedherein, refers to a molecule that may be expressed, released, secretedor delivered to a target by the modified cell described herein. Theagent includes, but is not limited to, a nucleic acid, an antibiotic, ananti-inflammatory agent, an antibody or antibody fragments thereof, agrowth factor, a cytokine, an enzyme, a protein, a peptide, a fusionprotein, a synthetic molecule, an organic molecule (e.g., a smallmolecule), a carbohydrate or the like, a lipid, a hormone, a microsome,a derivative or a variation thereof, and any combination thereof. Theagent may bind any cell moiety, such as a receptor, an antigenicdeterminant, or other binding site present on a target or target cell.The agent may diffuse or be transported into the cell, where it may actintracellularly.

The term “antibody,” as used herein, refers to an immunoglobulinmolecule which specifically binds with an antigen. Antibodies can beintact immunoglobulins derived from natural sources or from recombinantsources and can be immunoreactive portions of intact immunoglobulins.Antibodies are typically tetramers of immunoglobulin molecules. Theantibodies in the present invention may exist in a variety of formsincluding, for example, polyclonal antibodies, monoclonal antibodies,Fv, Fab and F(ab)₂, as well as single chain antibodies (scFv) andhumanized antibodies (Harlow et al., 1999, In: Using Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow etal., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor,N.Y.; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883;Bird et al., 1988, Science 242:423-426).

The term “antibody fragment” refers to a portion of an intact antibodyand refers to the antigenic determining variable regions of an intactantibody. Examples of antibody fragments include, but are not limitedto, Fab, Fab′, F(ab′)2, and Fv fragments, linear antibodies, scFvantibodies, and multispecific antibodies formed from antibody fragments.

An “antibody heavy chain,” as used herein, refers to the larger of thetwo types of polypeptide chains present in all antibody molecules intheir naturally occurring conformations.

An “antibody light chain,” as used herein, refers to the smaller of thetwo types of polypeptide chains present in all antibody molecules intheir naturally occurring conformations. α and β light chains refer tothe two major antibody light chain isotypes.

By the term “synthetic antibody” as used herein, is meant an antibodywhich is generated using recombinant DNA technology, such as, forexample, an antibody expressed by a bacteriophage as described herein.The term should also be construed to mean an antibody which has beengenerated by the synthesis of a DNA molecule encoding the antibody andwhich DNA molecule expresses an antibody protein, or an amino acidsequence specifying the antibody, wherein the DNA or amino acid sequencehas been obtained using synthetic DNA or amino acid sequence technologywhich is available and well known in the art.

The term “antigen” or “Ag” as used herein is defined as a molecule thatprovokes an immune response. This immune response may involve eitherantibody production, or the activation of specificimmunologically-competent cells, or both. The skilled artisan willunderstand that any macromolecule, including virtually all proteins orpeptides, can serve as an antigen. Furthermore, antigens can be derivedfrom recombinant or genomic DNA. A skilled artisan will understand thatany DNA, which comprises a nucleotide sequences or a partial nucleotidesequence encoding a protein that elicits an immune response thereforeencodes an “antigen” as that term is used herein. Furthermore, oneskilled in the art will understand that an antigen need not be encodedsolely by a full length nucleotide sequence of a gene. It is readilyapparent that the present invention includes, but is not limited to, theuse of partial nucleotide sequences of more than one gene and that thesenucleotide sequences are arranged in various combinations to elicit thedesired immune response. Moreover, a skilled artisan will understandthat an antigen need not be encoded by a “gene” at all. It is readilyapparent that an antigen can be generated synthesized or can be derivedfrom a biological sample. Such a biological sample can include, but isnot limited to a tissue sample, a tumor sample, a cell or a biologicalfluid.

The term “anti-tumor effect” as used herein, refers to a biologicaleffect which can be manifested by a decrease in tumor volume, a decreasein the number of tumor cells, a decrease in the number of metastases, anincrease in life expectancy, or amelioration of various physiologicalsymptoms associated with the cancerous condition. An “anti-tumor effect”can also be manifested by the ability of the peptides, polynucleotides,cells and antibodies of the invention in prevention of the occurrence oftumor in the first place.

The term “auto-antigen” means, in accordance with the present invention,any self-antigen which is recognized by the immune system as beingforeign. Auto-antigens comprise, but are not limited to, cellularproteins, phosphoproteins, cellular surface proteins, cellular lipids,nucleic acids, glycoproteins, including cell surface receptors.

The term “autoimmune disease” as used herein is defined as a disorderthat results from an autoimmune response. An autoimmune disease is theresult of an inappropriate and excessive response to a self-antigen.Examples of autoimmune diseases include but are not limited to,Addision's disease, alopecia areata, ankylosing spondylitis, autoimmunehepatitis, autoimmune parotitis, Crohn's disease, diabetes (Type I),dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis,Graves' disease, Guillain-Barr syndrome, Hashimoto's disease, hemolyticanemia, systemic lupus erythematosus, multiple sclerosis, myastheniagravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoidarthritis, sarcoidosis, scleroderma, Sjogren's syndrome,spondyloarthropathies, thyroiditis, vasculitis, vitiligo, myxedema,pernicious anemia, ulcerative colitis, among others.

As used herein, the term “autologous” is meant to refer to any materialderived from the same individual to which it is later to bere-introduced into the individual.

“Allogeneic” refers to a graft derived from a different animal of thesame species.

“Xenogeneic” refers to a graft derived from an animal of a differentspecies.

The term “cancer” as used herein is defined as disease characterized bythe rapid and uncontrolled growth of aberrant cells. Cancer cells canspread locally or through the bloodstream and lymphatic system to otherparts of the body. Examples of various cancers include but are notlimited to, breast cancer, prostate cancer, ovarian cancer, cervicalcancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer,liver cancer, brain cancer, lymphoma, leukemia, lung cancer and thelike. In certain embodiments, the cancer is medullary thyroid carcinoma.

The term “chimeric antigen receptor” or “CAR,” as used herein, refers toan artificial T cell surface receptor that is engineered to be expressedon an immune effector cell and specifically bind an antigen. CARS may beused as a therapy with adoptive cell transfer. Monocytes are removedfrom a patient (blood, tumor or ascites fluid) and modified so that theyexpress the receptors specific to a particular form of antigen. In someembodiments, the CARs have been expressed with specificity to a tumorassociated antigen, for example. CARs may also comprise an intracellularactivation domain, a transmembrane domain and an extracellular domaincomprising a tumor associated antigen binding region. In some aspects,CARs comprise fusions of single-chain variable fragments (scFv) derivedmonoclonal antibodies, fused to CD3-zeta transmembrane and intracellulardomain. The specificity of CAR designs may be derived from ligands ofreceptors (e.g., peptides). In some embodiments, a CAR can targetcancers by redirecting a monocyte/macrophage expressing the CAR specificfor tumor associated antigens.

The term “chimeric intracellular signaling molecule” refers torecombinant receptor comprising one or more intracellular domains of oneor more stimulatory and/or co-stimulatory molecules. The chimericintracellular signaling molecule substantially lacks an extracellulardomain. In some embodiments, the chimeric intracellular signalingmolecule comprises additional domains, such as a transmembrane domain, adetectable tag, and a spacer domain.

As used herein, the term “conservative sequence modifications” isintended to refer to amino acid modifications that do not significantlyaffect or alter the binding characteristics of the antibody containingthe amino acid sequence. Such conservative modifications include aminoacid substitutions, additions and deletions. Modifications can beintroduced into an antibody of the invention by standard techniquesknown in the art, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Conservative amino acid substitutions are ones in which theamino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine), beta-branchedside chains (e.g., threonine, valine, isoleucine) and aromatic sidechains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, oneor more amino acid residues within the CDR regions of an antibody can bereplaced with other amino acid residues from the same side chain familyand the altered antibody can be tested for the ability to bind antigensusing the functional assays described herein.

“Co-stimulatory ligand,” as the term is used herein, includes a moleculeon an antigen presenting cell (e.g., an aAPC, dendritic cell, B cell,and the like) that specifically binds a cognate co-stimulatory moleculeon a monocyte/macrophage, thereby providing a signal which mediates amonocyte/macrophageresponse, including, but not limited to,proliferation, activation, differentiation, and the like. Aco-stimulatory ligand can include, but is not limited to, CD7, B7-1(CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, induciblecostimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM),CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin betareceptor, 3/TR6, ILT3, ILT4, HVEM, an agonist or antibody that bindsToll ligand receptor and a ligand that specifically binds with B7-H3. Aco-stimulatory ligand also encompasses, inter alia, an antibody thatspecifically binds with a co-stimulatory molecule present on amonocyte/macrophage, such as, but not limited to, CD27, CD28, 4-1BB,OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1(LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specificallybinds with CD83.

A “co-stimulatory molecule” refers to a molecule on an innate immunecell that is used to heighten or dampen the initial stimulus. Forexample, pathogen-associated pattern recognition receptors, such as TLR(heighten) or the CD47/SIRPα axis (dampen), are molecules on innateimmune cells. Co-stimulatory molecules include, but are not limited toTCR, CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD86, common FcRgamma, FcR beta (Fc Epsilon Rib), CD79a, CD79b, Fcgamma RIIa, DAP10,DAP12, T cell receptor (TCR), CD27, CD28, 4-1BB (CD137), OX40, CD30,CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2,CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83,CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD127,CD160, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha,ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD,CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c,ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE/RANKL, DNAM1(CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9(CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A,Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162),LTBR, LAT, GADS, SLP-76, PAG/Cbp, NKp44, NKp30, NKp46, NKG2D, otherco-stimulatory molecules described herein, any derivative, variant, orfragment thereof, any synthetic sequence of a co-stimulatory moleculethat has the same functional capability, and any combination thereof.

A “co-stimulatory signal”, as used herein, refers to a signal, which incombination with a primary signal, such as activation of the CAR on amacrophage, leads to activation of the macrophage.

The term “cytotoxic” or “cytotoxicity” refers to killing or damagingcells. In one embodiment, cytotoxicity of the metabolically enhancedcells is improved, e.g. increased cytolytic activity of macrophages.

A “disease” is a state of health of an animal wherein the animal cannotmaintain homeostasis, and wherein if the disease is not ameliorated thenthe animal's health continues to deteriorate. In contrast, a “disorder”in an animal is a state of health in which the animal is able tomaintain homeostasis, but in which the animal's state of health is lessfavorable than it would be in the absence of the disorder. Leftuntreated, a disorder does not necessarily cause a further decrease inthe animal's state of health.

“Effective amount” or “therapeutically effective amount” are usedinterchangeably herein, and refer to an amount of a compound,formulation, material, or composition, as described herein effective toachieve a particular biological result or provides a therapeutic orprophylactic benefit. Such results may include, but are not limited to,anti-tumor activity as determined by any means suitable in the art.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA corresponding to thatgene produces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and thenon-coding strand, used as the template for transcription of a gene orcDNA, can be referred to as encoding the protein or other product ofthat gene or cDNA.

As used herein “endogenous” refers to any material from or producedinside an organism, cell, tissue or system.

As used herein, the term “exogenous” refers to any material introducedfrom or produced outside an organism, cell, tissue or system.

The term “expand” as used herein refers to increasing in number, as inan increase in the number of monocytes/macrophages. In one embodiment,the monocytes/macrophages that are expanded ex vivo increase in numberrelative to the number originally present in the culture. In anotherembodiment, the monocytes/macrophages that are expanded ex vivo increasein number relative to other cell types in the culture. The term “exvivo,” as used herein, refers to cells that have been removed from aliving organism, (e.g., a human) and propagated outside the organism(e.g., in a culture dish, test tube, or bioreactor).

The term “expression” as used herein is defined as the transcriptionand/or translation of a particular nucleotide sequence driven by itspromoter.

“Expression vector” refers to a vector comprising a recombinantpolynucleotide comprising expression control sequences operativelylinked to a nucleotide sequence to be expressed. An expression vectorcomprises sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host cell or in an in vitroexpression system. Expression vectors include all those known in theart, such as cosmids, plasmids (e.g., naked or contained in liposomes)and viruses (e.g., lentiviruses, retroviruses, adenoviruses, andadeno-associated viruses) that incorporate the recombinantpolynucleotide.

“Homologous” as used herein, refers to the subunit sequence identitybetween two polymeric molecules, e.g., between two nucleic acidmolecules, such as, two DNA molecules or two RNA molecules, or betweentwo polypeptide molecules. When a subunit position in both of the twomolecules is occupied by the same monomeric subunit; e.g., if a positionin each of two DNA molecules is occupied by adenine, then they arehomologous at that position. The homology between two sequences is adirect function of the number of matching or homologous positions; e.g.,if half (e.g., five positions in a polymer ten subunits in length) ofthe positions in two sequences are homologous, the two sequences are 50%homologous; if 90% of the positions (e.g., 9 of 10), are matched orhomologous, the two sequences are 90% homologous. As applied to thenucleic acid or protein, “homologous” as used herein refers to asequence that has about 50% sequence identity. More preferably, thehomologous sequence has about 75% sequence identity, even morepreferably, has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% sequence identity.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′)2 or other antigen-binding subsequences of antibodies)which contain minimal sequence derived from non-human immunoglobulin.For the most part, humanized antibodies are human immunoglobulins(recipient antibody) in which residues from a complementary-determiningregion (CDR) of the recipient are replaced by residues from a CDR of anon-human species (donor antibody) such as mouse, rat or rabbit havingthe desired specificity, affinity, and capacity. In some instances, Fvframework region (FR) residues of the human immunoglobulin are replacedby corresponding non-human residues. Furthermore, humanized antibodiescan comprise residues which are found neither in the recipient antibodynor in the imported CDR or framework sequences. These modifications aremade to further refine and optimize antibody performance. In general,the humanized antibody will comprise substantially all of at least one,and typically two, variable domains, in which all or substantially allof the CDR regions correspond to those of a non-human immunoglobulin andall or substantially all of the FR regions are those of a humanimmunoglobulin sequence. The humanized antibody optimally also willcomprise at least a portion of an immunoglobulin constant region (Fc),typically that of a human immunoglobulin. For further details, see Joneset al., Nature, 321: 522-525, 1986; Reichmann et al., Nature, 332:323-329, 1988; Presta, Curr. Op. Struct. Biol., 2: 593-596, 1992.

“Fully human” refers to an immunoglobulin, such as an antibody, wherethe whole molecule is of human origin or consists of an amino acidsequence identical to a human form of the antibody.

“Identity” as used herein refers to the subunit sequence identitybetween two polymeric molecules particularly between two amino acidmolecules, such as, between two polypeptide molecules. When two aminoacid sequences have the same residues at the same positions; e.g., if aposition in each of two polypeptide molecules is occupied by anArginine, then they are identical at that position. The identity orextent to which two amino acid sequences have the same residues at thesame positions in an alignment is often expressed as a percentage. Theidentity between two amino acid sequences is a direct function of thenumber of matching or identical positions; e.g., if half (e.g., fivepositions in a polymer ten amino acids in length) of the positions intwo sequences are identical, the two sequences are 50% identical; if 90%of the positions (e.g., 9 of 10), are matched or identical, the twoamino acids sequences are 90% identical.

By “substantially identical” is meant a polypeptide or nucleic acidmolecule exhibiting at least 50% identity to a reference amino acidsequence (for example, any one of the amino acid sequences describedherein) or nucleic acid sequence (for example, any one of the nucleicacid sequences described herein). Preferably, such a sequence is atleast 60%, more preferably 80% or 85%, and more preferably 90%, 95% oreven 99% identical at the amino acid level or nucleic acid to thesequence used for comparison.

The guide nucleic acid sequence may be complementary to one strand(nucleotide sequence) of a double stranded DNA target site. Thepercentage of complementation between the guide nucleic acid sequenceand the target sequence can be at least 50%, 51%, 52%, 53%, 54%, 55%,56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 63%, 65%, 66%, 67%, 68%, 69%,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or 100%. The guide nucleic acid sequence can be at least 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35 or more nucleotides in length. In someembodiments, the guide nucleic acid sequence comprises a contiguousstretch of 10 to 40 nucleotides. The variable targeting domain can becomposed of a DNA sequence, a RNA sequence, a modified DNA sequence, amodified RNA sequence (see for example modifications described herein),or any combination thereof.

Sequence identity is typically measured using sequence analysis software(for example, Sequence Analysis Software Package of the GeneticsComputer Group, University of Wisconsin Biotechnology Center, 1710University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, orPILEUP/PRETTYBOX programs). Such software matches identical or similarsequences by assigning degrees of homology to various substitutions,deletions, and/or other modifications. Conservative substitutionstypically include substitutions within the following groups: glycine,alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid,asparagine, glutamine; serine, threonine; lysine, arginine; andphenylalanine, tyrosine. In an exemplary approach to determining thedegree of identity, a BLAST program may be used, with a probabilityscore between e⁻³ and e⁻¹⁰⁰ indicating a closely related sequence.

The term “immunoglobulin” or “Ig,” as used herein is defined as a classof proteins, which function as antibodies. Antibodies expressed by Bcells are sometimes referred to as the BCR (B cell receptor) or antigenreceptor. The five members included in this class of proteins are IgA,IgG, IgM, IgD, and IgE. IgA is the primary antibody that is present inbody secretions, such as saliva, tears, breast milk, gastrointestinalsecretions and mucus secretions of the respiratory and genitourinarytracts. IgG is the most common circulating antibody. IgM is the mainimmunoglobulin produced in the primary immune response in most subjects.It is the most efficient immunoglobulin in agglutination, complementfixation, and other antibody responses, and is important in defenseagainst bacteria and viruses. IgD is the immunoglobulin that has noknown antibody function, but may serve as an antigen receptor. IgE isthe immunoglobulin that mediates immediate hypersensitivity by causingrelease of mediators from mast cells and basophils upon exposure toallergen.

The term “immune response” as used herein is defined as a cellularresponse to an antigen that occurs when lymphocytes identify antigenicmolecules as foreign and induce the formation of antibodies and/oractivate lymphocytes to remove the antigen.

As used herein, an “instructional material” includes a publication, arecording, a diagram, or any other medium of expression which can beused to communicate the usefulness of the compositions and methods ofthe invention. The instructional material of the kit of the inventionmay, for example, be affixed to a container which contains the nucleicacid, peptide, and/or composition of the invention or be shippedtogether with a container which contains the nucleic acid, peptide,and/or composition. Alternatively, the instructional material may beshipped separately from the container with the intention that theinstructional material and the compound be used cooperatively by therecipient.

“Isolated” means altered or removed from the natural state. For example,a nucleic acid or a peptide naturally present in a living animal is not“isolated,” but the same nucleic acid or peptide partially or completelyseparated from the coexisting materials of its natural state is“isolated.” An isolated nucleic acid or protein can exist insubstantially purified form, or can exist in a non-native environmentsuch as, for example, a host cell.

A “lentivirus” as used herein refers to a genus of the Retroviridaefamily. Lentiviruses are unique among the retroviruses in being able toinfect non-dividing cells; they can deliver a significant amount ofgenetic information into the DNA of the host cell, so they are one ofthe most efficient methods of a gene delivery vector. HIV, SIV, and FIVare all examples of lentiviruses. Vectors derived from lentivirusesoffer the means to achieve significant levels of gene transfer in vivo.

By the term “modified” as used herein, is meant a changed state orstructure of a molecule or cell of the invention. Molecules may bemodified in many ways, including chemically, structurally, andfunctionally. Cells may be modified through the introduction of nucleicacids.

By the term “modulating,” as used herein, is meant mediating adetectable increase or decrease in the level of a response in a subjectcompared with the level of a response in the subject in the absence of atreatment or compound, and/or compared with the level of a response inan otherwise identical but untreated subject. The term encompassesperturbing and/or affecting a native signal or response therebymediating a beneficial therapeutic response in a subject, preferably, ahuman.

In the context of the present invention, the following abbreviations forthe commonly occurring nucleic acid bases are used. “A” refers toadenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refersto thymidine, and “U” refers to uridine.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence. Thephrase nucleotide sequence that encodes a protein or an RNA may alsoinclude introns to the extent that the nucleotide sequence encoding theprotein may in some version contain an intron(s).

The term “operably linked” refers to functional linkage between aregulatory sequence and a heterologous nucleic acid sequence resultingin expression of the latter. For example, a first nucleic acid sequenceis operably linked with a second nucleic acid sequence when the firstnucleic acid sequence is placed in a functional relationship with thesecond nucleic acid sequence. For instance, a promoter is operablylinked to a coding sequence if the promoter affects the transcription orexpression of the coding sequence. Generally, operably linked DNAsequences are contiguous and, where necessary to join two protein codingregions, in the same reading frame.

The term “overexpressed” tumor antigen or “overexpression” of a tumorantigen is intended to indicate an abnormal level of expression of atumor antigen in a cell from a disease area like a solid tumor within aspecific tissue or organ of the patient relative to the level ofexpression in a normal cell from that tissue or organ. Patients havingsolid tumors or a hematological malignancy characterized byoverexpression of the tumor antigen can be determined by standard assaysknown in the art.

“Parenteral” administration of an immunogenic composition includes,e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.),intratumoral (i.t.) or intra-peritoneal (i.p.), or intrasternalinjection, or infusion techniques.

The term “polynucleotide” as used herein is defined as a chain ofnucleotides. Furthermore, nucleic acids are polymers of nucleotides.Thus, nucleic acids and polynucleotides as used herein areinterchangeable. One skilled in the art has the general knowledge thatnucleic acids are polynucleotides, which can be hydrolyzed into themonomeric “nucleotides.” The monomeric nucleotides can be hydrolyzedinto nucleosides. As used herein polynucleotides include, but are notlimited to, all nucleic acid sequences which are obtained by any meansavailable in the art, including, without limitation, recombinant means,i.e., the cloning of nucleic acid sequences from a recombinant libraryor a cell genome, using ordinary cloning technology and PCR™, and thelike, and by synthetic means.

As used herein, the terms “peptide,” “polypeptide,” and “protein” areused interchangeably, and refer to a compound comprised of amino acidresidues covalently linked by peptide bonds. A protein or peptide mustcontain at least two amino acids, and no limitation is placed on themaximum number of amino acids that can comprise a protein's or peptide'ssequence. Polypeptides include any peptide or protein comprising two ormore amino acids joined to each other by peptide bonds. As used herein,the term refers to both short chains, which also commonly are referredto in the art as peptides, oligopeptides and oligomers, for example, andto longer chains, which generally are referred to in the art asproteins, of which there are many types. “Polypeptides” include, forexample, biologically active fragments, substantially homologouspolypeptides, oligopeptides, homodimers, heterodimers, variants ofpolypeptides, modified polypeptides, derivatives, analogs, fusionproteins, among others. The polypeptides include natural peptides,recombinant peptides, synthetic peptides, or a combination thereof.

The term “promoter” as used herein is defined as a DNA sequencerecognized by the synthetic machinery of the cell, or introducedsynthetic machinery, required to initiate the specific transcription ofa polynucleotide sequence.

As used herein, the term “promoter/regulatory sequence” means a nucleicacid sequence which is required for expression of a gene productoperably linked to the promoter/regulatory sequence. In some instances,this sequence may be the core promoter sequence and in other instances,this sequence may also include an enhancer sequence and other regulatoryelements which are required for expression of the gene product. Thepromoter/regulatory sequence may, for example, be one which expressesthe gene product in a tissue specific manner.

A “constitutive” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a cell under most or allphysiological conditions of the cell.

An “inducible” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a cell substantially only whenan inducer which corresponds to the promoter is present in the cell.

A “tissue-specific” promoter is a nucleotide sequence which, whenoperably linked with a polynucleotide encodes or specified by a gene,causes the gene product to be produced in a cell substantially only ifthe cell is a cell of the tissue type corresponding to the promoter.

The term “resistance to immunosuppression” refers to lack of suppressionor reduced suppression of an immune system activity or activation.

A “signal transduction pathway” refers to the biochemical relationshipbetween a variety of signal transduction molecules that play a role inthe transmission of a signal from one portion of a cell to anotherportion of a cell. The phrase “cell surface receptor” includes moleculesand complexes of molecules capable of receiving a signal andtransmitting signal across the plasma membrane of a cell.

“Single chain antibodies” refer to antibodies formed by recombinant DNAtechniques in which immunoglobulin heavy and light chain fragments arelinked to the Fv region via an engineered span of amino acids. Variousmethods of generating single chain antibodies are known, including thosedescribed in U.S. Pat. No. 4,694,778; Bird (1988) Science 242:423-442;Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; Ward etal. (1989) Nature 334:54454; Skerra et al. (1988) Science 242:1038-1041.

By the term “specifically binds,” as used herein with respect to anantibody, is meant an antibody which recognizes a specific antigen, butdoes not substantially recognize or bind other molecules in a sample.For example, an antibody that specifically binds to an antigen from onespecies may also bind to that antigen from one or more species. But,such cross-species reactivity does not itself alter the classificationof an antibody as specific. In another example, an antibody thatspecifically binds to an antigen may also bind to different allelicforms of the antigen. However, such cross reactivity does not itselfalter the classification of an antibody as specific. In some instances,the terms “specific binding” or “specifically binding,” can be used inreference to the interaction of an antibody, a protein, or a peptidewith a second chemical species, to mean that the interaction isdependent upon the presence of a particular structure (e.g., anantigenic determinant or epitope) on the chemical species; for example,an antibody recognizes and binds to a specific protein structure ratherthan to proteins generally. If an antibody is specific for epitope “A”,the presence of a molecule containing epitope A (or free, unlabeled A),in a reaction containing labeled “A” and the antibody, will reduce theamount of labeled A bound to the antibody.

By the term “stimulation,” is meant a primary response induced bybinding of a stimulatory molecule (e.g., a TCR/CD3 complex) with itscognate ligand thereby mediating a signal transduction event, such as,but not limited to, signal transduction via the Fc receptor machinery orvia the synthetic CAR. Stimulation can mediate altered expression ofcertain molecules, such as downregulation of TGF-beta, and/orreorganization of cytoskeletal structures, and the like.

A “stimulatory molecule,” as the term is used herein, means a moleculeon a monocyte/macrophage that specifically binds with a cognatestimulatory ligand present on an antigen presenting cell.

A “stimulatory ligand,” as used herein, means a ligand that when presenton an antigen presenting cell (e.g., an aAPC, a dendritic cell, aB-cell, and the like) or tumor cell can specifically bind with a cognatebinding partner (referred to herein as a “stimulatory molecule”) on amonocyte/macrophage, thereby mediating a response by the immune cell,including, but not limited to, activation, initiation of an immuneresponse, proliferation, and the like. Stimulatory ligands arewell-known in the art and encompass, inter alia, Toll-like receptor(TLR) ligand, an anti-toll-like receptor antibody, an agonist, and anantibody for a monocyte/macrophage receptor. In addition, cytokines,such as interferon-gamma, are potent stimulants of macrophages.

The term “subject” is intended to include living organisms in which animmune response can be elicited (e.g., mammals). A “subject” or“patient,” as used therein, may be a human or non-human mammal.Non-human mammals include, for example, livestock and pets, such asovine, bovine, porcine, canine, feline and murine mammals. Preferably,the subject is human.

As used herein, a “substantially purified” cell is a cell that isessentially free of other cell types. A substantially purified cell alsorefers to a cell which has been separated from other cell types withwhich it is normally associated in its naturally occurring state. Insome instances, a population of substantially purified cells refers to ahomogenous population of cells. In other instances, this term referssimply to cell that have been separated from the cells with which theyare naturally associated in their natural state. In some embodiments,the cells are cultured in vitro. In other embodiments, the cells are notcultured in vitro.

A “target site” or “target sequence” refers to a genomic nucleic acidsequence that defines a portion of a nucleic acid to which a bindingmolecule may specifically bind under conditions sufficient for bindingto occur,

By “target” is meant a cell, organ, or site within the body that is inneed of treatment.

As used herein, the term “T cell receptor” or “TCR” refers to a complexof membrane proteins that participate in the activation of T cells inresponse to the presentation of antigen. The TCR is responsible forrecognizing antigens bound to major histocompatibility complexmolecules. TCR is composed of a heterodimer of an alpha (α) and beta (β)chain, although in some cells the TCR consists of gamma and delta (γ/δ)chains. TCRs may exist in alpha/beta and gamma/delta forms, which arestructurally similar but have distinct anatomical locations andfunctions. Each chain is composed of two extracellular domains, avariable and constant domain. In some embodiments, the TCR may bemodified on any cell comprising a TCR, including, for example, a helperI cell, a cytotoxic T cell, a memory T cell, regulatory T cell, naturalkiller I cell, and gamma delta T cell.

The term “therapeutic” as used herein means a treatment and/orprophylaxis. A therapeutic effect is obtained by suppression, remission,or eradication of a disease state.

The term “transfected” or “transformed” or “transduced” as used hereinrefers to a process by which exogenous nucleic acid is transferred orintroduced into the host cell. A “transfected” or “transformed” or“transduced” cell is one which has been transfected, transformed ortransduced with exogenous nucleic acid. The cell includes the primarysubject cell and its progeny.

To “treat” a disease as the term is used herein, means to reduce thefrequency or severity of at least one sign or symptom of a disease ordisorder experienced by a subject.

The term “tumor” as used herein, refers to an abnormal growth of tissuethat may be benign, pre-cancerous, malignant, or metastatic.

The phrase “under transcriptional control” or “operatively linked” asused herein means that the promoter is in the correct location andorientation in relation to a polynucleotide to control the initiation oftranscription by RNA polymerase and expression of the polynucleotide.

A “vector” is a composition of matter which comprises an isolatednucleic acid and which can be used to deliver the isolated nucleic acidto the interior of a cell. Numerous vectors are known in the artincluding, but not limited to, linear polynucleotides, polynucleotidesassociated with ionic or amphiphilic compounds, plasmids, and viruses.Thus, the term “vector” includes an autonomously replicating plasmid ora virus. The term should also be construed to include non-plasmid andnon-viral compounds which facilitate transfer of nucleic acid intocells, such as, for example, polylysine compounds, liposomes, and thelike. Examples of viral vectors include, but are not limited to,adenoviral vectors, adeno-associated virus vectors, retroviral vectors,lentiviral vectors, and the like.

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

DESCRIPTION

Accumulating evidence suggests that macrophages are abundant in thetumor microenvironment of numerous cancers where they can adopt aclassically activated (M1, antitumor) or an alternatively activated (M2,pro-tumor) phenotype. Macrophages are potent effectors of the innateimmune system and are capable of at least three distinct anti-tumorfunctions: phagocytosis, cellular cytotoxicity, and antigen presentationto orchestrate an adaptive immune response. While T cells requireantigen-dependent activation via the T cell receptor or the chimericimmunoreceptor, macrophages can be activated in a variety of ways.Direct macrophage activation is antigen-independent, relying onmechanisms such as pathogen associated molecular pattern recognition byToll-like receptors (TLRs). Immune-complex mediated activation isantigen dependent but requires the presence of antigen-specificantibodies and absence of the inhibitory CD47-SIRPα interaction.

Tumor-associated macrophages have been shown to be re-programmable bythe tumor microenvironment to become key immunosuppressive players inthe microenvironment. Therefore, the ability to genetically engineermacrophages to prevent the development of an immunosuppressive geneticreprogram would represent a vertical advance in the field.

The present invention includes compositions and methods for treating amalignancy in a subject. The invention includes expression of a chimericantigen receptor in a monocyte, macrophage or dendritic cell. Such amodified cell is recruited to the tumor microenvironment where it actsas a potent immune effector by infiltrating the tumor and killing targetcells.

Chimeric Antigen Receptor (CAR)

In one aspect of the invention, a modified monocyte, macrophage, ordendritic cell is generated by expressing a CAR therein. Thus, thepresent invention encompasses a CAR and a nucleic acid constructencoding a CAR, wherein the CAR includes an antigen binding domain, atransmembrane domain and an intracellular domain.

In one aspect, the invention includes a modified cell comprising achimeric antigen receptor (CAR), wherein the CAR comprises an antigenbinding domain, a transmembrane domain and an intracellular domain of aco-stimulatory molecule, and wherein cell is a monocyte, macrophage, ordendritic cell that possesses targeted effector activity. In anotheraspect, the invention includes a modified cell comprising a nucleic acidsequence encoding a chimeric antigen receptor (CAR), wherein nucleicacid sequence comprises a nucleic acid sequence encoding an antigenbinding domain, a nucleic acid sequence encoding a transmembrane domainand a nucleic acid sequence encoding an intracellular domain of aco-stimulatory molecule, and wherein the cell is a monocyte, macrophage,or dendritic cell that expresses the CAR and possesses targeted effectoractivity. In one embodiment, the targeted effector activity is directedagainst an antigen on a target cell that specifically binds the antigenbinding domain of the CAR. In another embodiment, the targeted effectoractivity is selected from the group consisting of phagocytosis, targetedcellular cytotoxicity, antigen presentation, and cytokine secretion.

Antigen Binding Domain

In one embodiment, the CAR of the invention comprises an antigen bindingdomain that binds to an antigen on a target cell. Examples of cellsurface markers that may act as an antigen that binds to the antigenbinding domain of the CAR include those associated with viral, bacterialand parasitic infections, autoimmune disease, and cancer cells.

The choice of antigen binding domain depends upon the type and number ofantigens that are present on the surface of a target cell. For example,the antigen binding domain may be chosen to recognize an antigen thatacts as a cell surface marker on a target cell associated with aparticular disease state.

In one embodiment, the antigen binding domain binds to a tumor antigen,such as an antigen that is specific for a tumor or cancer of interest.In one embodiment, the tumor antigen of the present invention comprisesone or more antigenic cancer epitopes. Nonlimiting examples of tumorassociated antigens include CD19; CD123; CD22; CD30; CD171; CS-1 (alsoreferred to as CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24); C-typelectin-like molecule-1 (CLL-1 or CLECL1); CD33; epidermal growth factorreceptor variant III (EGFRvIII); ganglioside G2 (GD2); ganglioside GD3(aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); TNF receptor familymember B cell maturation (BCMA); Tn antigen ((Tn Ag) or(GalNAcα-Ser/Thr)); prostate-specific membrane antigen (PSMA); Receptortyrosine kinase-like orphan receptor 1 (ROR1); Fms-Like Tyrosine Kinase3 (FLT3); Tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6;Carcinoembryonic antigen (CEA); Epithelial cell adhesion molecule(EPCAM); B7H3 (CD276); KIT (CD117); Interleukin-13 receptor subunitalpha-2 (IL-13Ra2 or CD213A2); Mesothelin; Interleukin 11 receptor alpha(IL-11Ra); prostate stem cell antigen (PSCA); Protease Serine 21(Testisin or PRSS21); vascular endothelial growth factor receptor 2(VEGFR2); Lewis(Y) antigen; CD24; Platelet-derived growth factorreceptor beta (PDGFR-beta); Stage-specific embryonic antigen-4 (SSEA-4);CD20; Folate receptor alpha; Receptor tyrosine-protein kinase ERBB2(Her2/neu); Mucin 1, cell surface associated (MUC1); epidermal growthfactor receptor (EGFR); neural cell adhesion molecule (NCAM); Prostase;prostatic acid phosphatase (PAP); elongation factor 2 mutated (ELF2M);Ephrin B2; fibroblast activation protein alpha (FAP); insulin-likegrowth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX);Proteasome (Prosome, Macropain) Subunit, Beta Type, 9 (LMP2);glycoprotein 100 (gp100); oncogene fusion protein consisting ofbreakpoint cluster region (BCR) and Abelson murine leukemia viraloncogene homolog 1 (Abl) (bcr-abl); tyrosinase; ephrin type-A receptor 2(EphA2); Fucosyl GM1; sialyl Lewis adhesion molecule (sLe); gangliosideGM3 (aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); transglutaminase 5 (TGS5);high molecular weight-melanoma-associated antigen (HMWMAA); o-acetyl-GD2ganglioside (OAcGD2); Folate receptor beta; tumor endothelial marker 1(TEM1/CD248); tumor endothelial marker 7-related (TEM7R); claudin 6(CLDN6); thyroid stimulating hormone receptor (TSHR); G protein-coupledreceptor class C group 5, member D (GPRC5D); chromosome X open readingframe 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK);Polysialic acid; placenta-specific 1 (PLAC1); hexasaccharide portion ofgloboH glycoceramide (GloboH); mammary gland differentiation antigen(NY-BR-1); uroplakin 2 (UPK2); Hepatitis A virus cellular receptor 1(HAVCR1); adrenoceptor beta 3 (ADRB3); pannexin 3 (PANX3); Gprotein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex, locusK 9 (LY6K); Olfactory receptor 51E2 (OR51E2); TCR Gamma AlternateReading Frame Protein (TARP); Wilms tumor protein (WT1); Cancer/testisantigen 1 (NY-ESO-1); Cancer/testis antigen 2 (LAGE-1a);Melanoma-associated antigen 1 (MAGE-A1); ETS translocation-variant gene6, located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); XAntigen Family, Member 1A (XAGE1); angiopoietin-binding cell surfacereceptor 2 (Tie 2); melanoma cancer testis antigen-1 (MAD-CT-1);melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1;tumor protein p53 (p53); p53 mutant; prostein; surviving; telomerase;prostate carcinoma tumor antigen-1 (PCTA-1 or Galectin 8), melanomaantigen recognized by T cells 1 (MelanA or MART1); Rat sarcoma (Ras)mutant; human Telomerase reverse transcriptase (hTERT); sarcomatranslocation breakpoints; melanoma inhibitor of apoptosis (ML-IAP); ERG(transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-Acetylglucosaminyl-transferase V (NA17); paired box protein Pax-3 (PAX3);Androgen receptor; Cyclin B1; v-myc avian myelocytomatosis viraloncogene neuroblastoma derived homolog (MYCN); Ras Homolog Family MemberC (RhoC); Tyrosinase-related protein 2 (TRP-2); Cytochrome P450 1B1(CYP1B1); CCCTC-Binding Factor (Zinc Finger Protein)-Like (BORIS orBrother of the Regulator of Imprinted Sites), Squamous Cell CarcinomaAntigen Recognized By T Cells 3 (SART3); Paired box protein Pax-5(PAX5); proacrosin binding protein sp32 (OY-TES1); lymphocyte-specificprotein tyrosine kinase (LCK); A kinase anchor protein 4 (AKAP-4);synovial sarcoma, X breakpoint 2 (SSX2); Receptor for Advanced GlycationEndproducts (RAGE-1); renal ubiquitous 1 (RU1); renal ubiquitous 2(RU2); legumain; human papilloma virus E6 (HPV E6); human papillomavirus E7 (HPV E7); intestinal carboxyl esterase; heat shock protein 70-2mutated (mut hsp70-2); CD79a; CD79b; CD72; Leukocyte-associatedimmunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor(FCAR or CD89); Leukocyte immunoglobulin-like receptor subfamily Amember 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-typelectin domain family 12 member A (CLEC12A); bone marrow stromal cellantigen 2 (BST2); EGF-like module-containing mucin-like hormonereceptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); Glypican-3 (GPC3);Fc receptor-like 5 (FCRL5); and immunoglobulin lambda-like polypeptide 1(IGLL1).

The antigen binding domain can include any domain that binds to theantigen and may include, but is not limited to, a monoclonal antibody, apolyclonal antibody, a synthetic antibody, a human antibody, a humanizedantibody, a non-human antibody, and any fragment thereof. Thus, in oneembodiment, the antigen binding domain portion comprises a mammalianantibody or a fragment thereof. In another embodiment, the antigenbinding domain of the CAR is selected from the group consisting of ananti-CD19 antibody, an anti-HER2 antibody, and a fragment thereof.

In some instances, the antigen binding domain is derived from the samespecies in which the CAR will ultimately be used in. For example, foruse in humans, it the antigen binding domain of the CAR comprises ahuman antibody, a humanized antibody, or a fragment thereof.

In some aspects of the invention, the antigen binding domain is operablylinked to another domain of the CAR, such as the transmembrane domain orthe intracellular domain, for expression in the cell. In one embodiment,a nucleic acid encoding the antigen binding domain is operably linked toa nucleic acid encoding a transmembrane domain and a nucleic acidencoding an intracellular domain.

Transmembrane Domain

With respect to the transmembrane domain, the CAR can be designed tocomprise a transmembrane domain that connects the antigen binding domainof the CAR to the intracellular domain. In one embodiment, thetransmembrane domain is naturally associated with one or more of thedomains in the CAR. In some instances, the transmembrane domain can beselected or modified by amino acid substitution to avoid binding of suchdomains to the transmembrane domains of the same or different surfacemembrane proteins to minimize interactions with other members of thereceptor complex.

The transmembrane domain may be derived either from a natural or from asynthetic source. Where the source is natural, the domain may be derivedfrom any membrane-bound or transmembrane protein. Transmembrane regionsof particular use in this invention may be derived from (i.e. compriseat least the transmembrane region(s) of) the alpha, beta or zeta chainof the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9,CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, Toll-likereceptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, and TLR9.In some instances, a variety of human hinges can be employed as wellincluding the human Ig (immunoglobulin) hinge.

In one embodiment, the transmembrane domain may be synthetic, in whichcase it will comprise predominantly hydrophobic residues such as leucineand valine. Preferably a triplet of phenylalanine, tryptophan and valinewill be found at each end of a synthetic transmembrane domain.

Intracellular Domain

The intracellular domain or otherwise, the cytoplasmic domain of theCAR, includes a similar or the same intracellular domain as the chimericintracellular signaling molecule described elsewhere herein, and isresponsible for activation of the cell in which the CAR is expressed.

In one embodiment, the intracellular domain of the CAR includes a domainresponsible for signal activation and/or transduction.

Examples of an intracellular domain for use in the invention include,but are not limited to, the cytoplasmic portion of a surface receptor,co-stimulatory molecule, and any molecule that acts in concert toinitiate signal transduction in the monocyte, macrophage or dendriticcell, as well as any derivative or variant of these elements and anysynthetic sequence that has the same functional capability.

Examples of the intracellular domain include a fragment or domain fromone or more molecules or receptors including, but are not limited to,TCR, CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD86, common FcRgamma, FcR beta (Fc Epsilon Rib), CD79a, CD79b, Fcgamma RIIa, DAP10,DAP12, T cell receptor (TCR), CD27, CD28, 4-1BB (CD137), OX40, CD30,CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2,CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83,CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD127,CD160, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha,ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD,CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c,ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE/RANKL, DNAM1(CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9(CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A,Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162),LTBR, LAT, GADS, SLP-76, PAG/Cbp, NKp44, NKp30, NKp46, NKG2D, Toll-likereceptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, otherco-stimulatory molecules described herein, any derivative, variant, orfragment thereof, any synthetic sequence of a co-stimulatory moleculethat has the same functional capability, and any combination thereof.

In one embodiment, the intracellular domain of the CAR comprises dualsignaling domains, such as 41BB, CD28, ICOS, TLR1, TLR2, TLR3, TLR4,TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, CD116 receptor beta chain,CSF1-R, LRP1/CD91, SR-A1, SR-A2, MARCO, SR-CL1, SR-CL2, SR-C, SR-E, CR1,CR3, CR4, dectin 1, DEC-205, DC-SIGN, CD14, CD36, LOX-1, CD11b, togetherwith any of the signaling domains listed in the above paragraph in anycombination. In another embodiment, the intracellular domain of the CARincludes any portion of one or more co-stimulatory molecules, such as atleast one signaling domain from CD3, Fc epsilon RI gamma chain, anyderivative or variant thereof, any synthetic sequence thereof that hasthe same functional capability, and any combination thereof.

Between the antigen binding domain and the transmembrane domain of theCAR, or between the intracellular domain and the transmembrane domain ofthe CAR, a spacer domain may be incorporated. As used herein, the term“spacer domain” generally means any oligo- or polypeptide that functionsto link the transmembrane domain to, either the antigen binding domainor, the intracellular domain in the polypeptide chain. In oneembodiment, the spacer domain may comprise up to 300 amino acids,preferably 10 to 100 amino acids and most preferably 25 to 50 aminoacids. In another embodiment, a short oligo- or polypeptide linker,preferably between 2 and 10 amino acids in length may form the linkagebetween the transmembrane domain and the intracellular domain of theCAR. An example of a linker includes a glycine-serine doublet.

Human Antibodies

It may be preferable to use human antibodies or fragments thereof whenusing the antigen binding domain of a CAR. Completely human antibodiesare particularly desirable for therapeutic treatment of human subjects.Human antibodies can be made by a variety of methods known in the artincluding phage display methods using antibody libraries derived fromhuman immunoglobulin sequences, including improvements to thesetechniques. See, also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCTpublications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO96/34096, WO 96/33735, and WO 91/10741; each of which is incorporatedherein by reference in its entirety.

Human antibodies can also be produced using transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichcan express human immunoglobulin genes. For example, the human heavy andlight chain immunoglobulin gene complexes may be introduced randomly orby homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion may be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes may be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. For example, it has been described that thehomozygous deletion of the antibody heavy chain joining region (JH) genein chimeric and germ-line mutant mice results in complete inhibition ofendogenous antibody production. The modified embryonic stem cells areexpanded and microinjected into blastocysts to produce chimeric mice.The chimeric mice are then bred to produce homozygous offspring whichexpress human antibodies. The transgenic mice are immunized in thenormal fashion with a selected antigen, e.g., all or a portion of apolypeptide of the invention. Antibodies directed against the target ofchoice can be obtained from the immunized, transgenic mice usingconventional hybridoma technology. The human immunoglobulin transgenesharbored by the transgenic mice rearrange during B cell differentiation,and subsequently undergo class switching and somatic mutation. Thus,using such a technique, it is possible to produce therapeutically usefulIgG, IgA, IgM and IgE antibodies, including, but not limited to, IgG1(gamma 1) and IgG3. For an overview of this technology for producinghuman antibodies, see, Lonberg and Huszar (Int. Rev. Immunol., 13:65-93(1995)). For a detailed discussion of this technology for producinghuman antibodies and human monoclonal antibodies and protocols forproducing such antibodies, see, e.g., PCT Publication Nos. WO 98/24893,WO 96/34096, and WO 96/33735; and U.S. Pat. Nos. 5,413,923; 5,625,126;5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; and 5,939,598,each of which is incorporated by reference herein in their entirety. Inaddition, companies such as Abgenix, Inc. (Freemont, Calif.) andGenpharm (San Jose, Calif.) can be engaged to provide human antibodiesdirected against a selected antigen using technology similar to thatdescribed above. For a specific discussion of transfer of a humangerm-line immunoglobulin gene array in germ-line mutant mice that willresult in the production of human antibodies upon antigen challenge see,e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993);Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann et al., Yearin Immunol., 7:33 (1993); and Duchosal et al., Nature, 355:258 (1992).

Human antibodies can also be derived from phage-display libraries(Hoogenboom et al., J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol.Biol., 222:581-597 (1991); Vaughan et al., Nature Biotech., 14:309(1996)). Phage display technology (McCafferty et al., Nature,348:552-553 (1990)) can be used to produce human antibodies and antibodyfragments in vitro, from immunoglobulin variable (V) domain generepertoires from unimmunized donors. According to this technique,antibody V domain genes are cloned in-frame into either a major or minorcoat protein gene of a filamentous bacteriophage, such as M13 or fd, anddisplayed as functional antibody fragments on the surface of the phageparticle. Because the filamentous particle contains a single-strandedDNA copy of the phage genome, selections based on the functionalproperties of the antibody also result in selection of the gene encodingthe antibody exhibiting those properties. Thus, the phage mimics some ofthe properties of the B cell. Phage display can be performed in avariety of formats; for their review see, e.g., Johnson, Kevin S, andChiswell, David J., Current Opinion in Structural Biology 3:564-571(1993). Several sources of V-gene segments can be used for phagedisplay. Clackson et al., Nature, 352:624-628 (1991) isolated a diversearray of anti-oxazolone antibodies from a small random combinatoriallibrary of V genes derived from the spleens of unimmunized mice. Arepertoire of V genes from unimmunized human donors can be constructedand antibodies to a diverse array of antigens (including self-antigens)can be isolated essentially following the techniques described by Markset al., J. Mol. Biol., 222:581-597 (1991), or Griffith et al., EMBO J.,12:725-734 (1993). See, also, U.S. Pat. Nos. 5,565,332 and 5,573,905,each of which is incorporated herein by reference in its entirety.

Human antibodies may also be generated by in vitro activated B cells(see, U.S. Pat. Nos. 5,567,610 and 5,229,275, each of which isincorporated herein by reference in its entirety). Human antibodies mayalso be generated in vitro using hybridoma techniques such as, but notlimited to, that described by Roder et al. (Methods Enzymol.,121:140-167 (1986)).

Humanized Antibodies

Alternatively, in some embodiments, a non-human antibody can behumanized, where specific sequences or regions of the antibody aremodified to increase similarity to an antibody naturally produced in ahuman. For instance, in the present invention, the antibody or fragmentthereof may comprise a non-human mammalian scFv. In one embodiment, theantigen binding domain portion is humanized.

A humanized antibody can be produced using a variety of techniques knownin the art, including but not limited to, CDR-grafting (see, e.g.,European Patent No. EP 239,400; International Publication No. WO91/09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089, eachof which is incorporated herein in its entirety by reference), veneeringor resurfacing (see, e.g., European Patent Nos. EP 592,106 and EP519,596; Padlan, 1991, Molecular Immunology, 28(4/5):489-498; Studnickaet al., 1994, Protein Engineering, 7(6):805-814; and Roguska et al.,1994, PNAS, 91:969-973, each of which is incorporated herein by itsentirety by reference), chain shuffling (see, e.g., U.S. Pat. No.5,565,332, which is incorporated herein in its entirety by reference),and techniques disclosed in, e.g., U.S. Patent Application PublicationNo. US2005/0042664, U.S. Patent Application Publication No.US2005/0048617, U.S. Pat. Nos. 6,407,213, 5,766,886, InternationalPublication No. WO 9317105, Tan et al., J. Immunol., 169:1119-25 (2002),Caldas et al., Protein Eng., 13(5):353-60 (2000), Morea et al., Methods,20(3):267-79 (2000), Baca et al., J. Biol. Chem., 272(16):10678-84(1997), Roguska et al., Protein Eng., 9(10):895-904 (1996), Couto etal., Cancer Res., 55 (23 Supp):5973s-5977s (1995), Couto et al., CancerRes., 55(8):1717-22 (1995), Sandhu J S, Gene, 150(2):409-10 (1994), andPedersen et al., J. Mol. Biol., 235(3):959-73 (1994), each of which isincorporated herein in its entirety by reference. Often, frameworkresidues in the framework regions will be substituted with thecorresponding residue from the CDR donor antibody to alter, preferablyimprove, antigen binding. These framework substitutions are identifiedby methods well-known in the art, e.g., by modeling of the interactionsof the CDR and framework residues to identify framework residuesimportant for antigen binding and sequence comparison to identifyunusual framework residues at particular positions. (See, e.g., Queen etal., U.S. Pat. No. 5,585,089; and Riechmann et al., 1988, Nature,332:323, which are incorporated herein by reference in theirentireties.)

A humanized antibody has one or more amino acid residues introduced intoit from a source which is nonhuman. These nonhuman amino acid residuesare often referred to as “import” residues, which are typically takenfrom an “import” variable domain. Thus, humanized antibodies compriseone or more CDRs from nonhuman immunoglobulin molecules and frameworkregions from human. Humanization of antibodies is well-known in the artand can essentially be performed following the method of Winter andco-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al.,Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536(1988)), by substituting rodent CDRs or CDR sequences for thecorresponding sequences of a human antibody, i.e., CDR-grafting (EP239,400; PCT Publication No. WO 91/09967; and U.S. Pat. Nos. 4,816,567;6,331,415; 5,225,539; 5,530,101; 5,585,089; 6,548,640, the contents ofwhich are incorporated herein by reference herein in their entirety). Insuch humanized chimeric antibodies, substantially less than an intacthuman variable domain has been substituted by the corresponding sequencefrom a nonhuman species. In practice, humanized antibodies are typicallyhuman antibodies in which some CDR residues and possibly some framework(FR) residues are substituted by residues from analogous sites in rodentantibodies. Humanization of antibodies can also be achieved by veneeringor resurfacing (EP 592,106; EP 519,596; Padlan, 1991, MolecularImmunology, 28(4/5):489-498; Studnicka et al., Protein Engineering,7(6):805-814 (1994); and Roguska et al., PNAS, 91:969-973 (1994)) orchain shuffling (U.S. Pat. No. 5,565,332), the contents of which areincorporated herein by reference herein in their entirety.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is to reduce antigenicity. Accordingto the so-called “best-fit” method, the sequence of the variable domainof a rodent antibody is screened against the entire library of knownhuman variable-domain sequences. The human sequence which is closest tothat of the rodent is then accepted as the human framework (FR) for thehumanized antibody (Sims et al., J. Immunol., 151:2296 (1993); Chothiaet al., J. Mol. Biol., 196:901 (1987), the contents of which areincorporated herein by reference herein in their entirety). Anothermethod uses a particular framework derived from the consensus sequenceof all human antibodies of a particular subgroup of light or heavychains. The same framework may be used for several different humanizedantibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992);Presta et al., J. Immunol., 151:2623 (1993), the contents of which areincorporated herein by reference herein in their entirety).

Antibodies can be humanized that retain high affinity for the targetantigen and that possess other favorable biological properties.According to one aspect of the invention, humanized antibodies areprepared by a process of analysis of the parental sequences and variousconceptual humanized products using three-dimensional models of theparental and humanized sequences. Three-dimensional immunoglobulinmodels are commonly available and are familiar to those skilled in theart. Computer programs are available which illustrate and displayprobable three-dimensional conformational structures of selectedcandidate immunoglobulin sequences. Inspection of these displays permitsanalysis of the likely role of the residues in the functioning of thecandidate immunoglobulin sequence, i.e., the analysis of residues thatinfluence the ability of the candidate immunoglobulin to bind the targetantigen. In this way, FR residues can be selected and combined from therecipient and import sequences so that the desired antibodycharacteristic, such as increased affinity for the target antigen, isachieved. In general, the CDR residues are directly and mostsubstantially involved in influencing antigen binding.

A humanized antibody retains a similar antigenic specificity as theoriginal antibody. However, using certain methods of humanization, theaffinity and/or specificity of binding of the antibody to the targetantigen may be increased using methods of “directed evolution,” asdescribed by Wu et al., J. Mol. Biol., 294:151 (1999), the contents ofwhich are incorporated herein by reference herein in their entirety.

Vectors

A vector may be used to introduce the CAR into a monocyte, macrophage ordendritic cell as described elsewhere herein. In one aspect, theinvention includes a vector comprising a nucleic acid sequence encodinga CAR as described herein. In one embodiment, the vector comprises aplasmid vector, viral vector, retrotransposon (e.g. piggyback, sleepingbeauty), site directed insertion vector (e.g. CRISPR, Zn fingernucleases, TALEN), or suicide expression vector, or other known vectorin the art.

All constructs mentioned above are capable of use with 3rd generationlentiviral vector plasmids, other viral vectors, or RNA approved for usein human cells. In one embodiment, the vector is a viral vector, such asa lentiviral vector. In another embodiment, the vector is a RNA vector.

The production of any of the molecules described herein can be verifiedby sequencing. Expression of the full length proteins may be verifiedusing immunoblot, immunohistochemistry, flow cytometry or othertechnology well known and available in the art.

The present invention also provides a vector in which DNA of the presentinvention is inserted. Vectors, including those derived fromretroviruses such as lentivirus, are suitable tools to achieve long-termgene transfer since they allow long-term, stable integration of atransgene and its propagation in daughter cells. Lentiviral vectors havethe added advantage over vectors derived from onco-retroviruses, such asmurine leukemia viruses, in that they can transduce non-proliferatingcells, such as hepatocytes. They also have the added advantage ofresulting in low immunogenicity in the subject into which they areintroduced.

The expression of natural or synthetic nucleic acids is typicallyachieved by operably linking a nucleic acid or portions thereof to apromoter, and incorporating the construct into an expression vector. Thevector is one generally capable of replication in a mammalian cell,and/or also capable of integration into the cellular genome of themammal. Typical vectors contain transcription and translationterminators, initiation sequences, and promoters useful for regulationof the expression of the desired nucleic acid sequence.

The nucleic acid can be cloned into any number of different types ofvectors. For example, the nucleic acid can be cloned into a vectorincluding, but not limited to a plasmid, a phagemid, a phage derivative,an animal virus, and a cosmid. Vectors of particular interest includeexpression vectors, replication vectors, probe generation vectors, andsequencing vectors.

The expression vector may be provided to a cell in the form of a viralvector. Viral vector technology is well known in the art and isdescribed, for example, in Sambrook et al., 2012, MOLECULAR CLONING: ALABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, NY), and inother virology and molecular biology manuals. Viruses, which are usefulas vectors include, but are not limited to, retroviruses, adenoviruses,adeno-associated viruses, herpes viruses, and lentiviruses. In general,a suitable vector contains an origin of replication functional in atleast one organism, a promoter sequence, convenient restrictionendonuclease sites, and one or more selectable markers, (e.g., WO01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).

Additional promoter elements, e.g., enhancers, regulate the frequency oftranscriptional initiation. Typically, these are located in the region30-110 bp upstream of the start site, although a number of promotershave recently been shown to contain functional elements downstream ofthe start site as well. The spacing between promoter elements frequentlyis flexible, so that promoter function is preserved when elements areinverted or moved relative to one another. In the thymidine kinase (tk)promoter, the spacing between promoter elements can be increased to 50bp apart before activity begins to decline. Depending on the promoter,it appears that individual elements can function either cooperatively orindependently to activate transcription.

An example of a promoter is the immediate early cytomegalovirus (CMV)promoter sequence. This promoter sequence is a strong constitutivepromoter sequence capable of driving high levels of expression of anypolynucleotide sequence operatively linked thereto. However, otherconstitutive promoter sequences may also be used, including, but notlimited to the simian virus 40 (SV40) early promoter, mouse mammarytumor virus (MMTV), human immunodeficiency virus (HIV) long terminalrepeat (LTR) promoter, MoMuLV promoter, an avian leukemia viruspromoter, an Epstein-Barr virus immediate early promoter, a Rous sarcomavirus promoter, the elongation factor-la promoter, as well as human genepromoters such as, but not limited to, the actin promoter, the myosinpromoter, the hemoglobin promoter, and the creatine kinase promoter.Further, the invention should not be limited to the use of constitutivepromoters. Inducible promoters are also contemplated as part of theinvention. The use of an inducible promoter provides a molecular switchcapable of turning on expression of the polynucleotide sequence which itis operatively linked when such expression is desired, or turning offthe expression when expression is not desired. Examples of induciblepromoters include, but are not limited to a metallothionine promoter, aglucocorticoid promoter, a progesterone promoter, and a tetracyclinepromoter.

In order to assess expression of a polypeptide or portions thereof, theexpression vector to be introduced into a cell can also contain either aselectable marker gene or a reporter gene or both to facilitateidentification and selection of expressing cells from the population ofcells sought to be transfected or infected through viral vectors. Inother aspects, the selectable marker may be carried on a separate pieceof DNA and used in a co-transfection procedure. Both selectable markersand reporter genes may be flanked with appropriate regulatory sequencesto enable expression in the host cells. Useful selectable markersinclude, for example, antibiotic-resistance genes, such as neo and thelike.

Reporter genes are used for identifying potentially transfected cellsand for evaluating the functionality of regulatory sequences. Ingeneral, a reporter gene is a gene that is not present in or expressedby the recipient organism or tissue and that encodes a polypeptide whoseexpression is manifested by some easily detectable property, e.g.,enzymatic activity. Expression of the reporter gene is assessed at asuitable time after the DNA has been introduced into the recipientcells. Suitable reporter genes may include genes encoding luciferase,beta-galactosidase, chloramphenicol acetyl transferase, secretedalkaline phosphatase, or the green fluorescent protein gene (e.g.,Ui-Tei et al., 2000 FEBS Letters 479: 79-82). Suitable expressionsystems are well known and may be prepared using known techniques orobtained commercially. In general, the construct with the minimal 5′flanking region showing the highest level of expression of reporter geneis identified as the promoter. Such promoter regions may be linked to areporter gene and used to evaluate agents for the ability to modulatepromoter-driven transcription.

Introduction of Nucleic Acids

In one aspect, the invention includes a method for modifying a cellcomprising introducing a chimeric antigen receptor (CAR) into themonocyte, macrophage, or dendritic cell, wherein the CAR comprises anantigen binding domain, a transmembrane domain and an intracellulardomain of a co-stimulatory molecule, and wherein the cell is a monocyte,macrophage, or dendritic cell that expresses the CAR and possessestargeted effector activity. In one embodiment, introducing the CAR intothe cell comprises introducing a nucleic acid sequence encoding the CAR.In another embodiment, introducing the nucleic acid sequence compriseselectroporating a mRNA encoding the CAR.

Methods of introducing and expressing genes, such as the CAR, into acell are known in the art. In the context of an expression vector, thevector can be readily introduced into a host cell, e.g., mammalian,bacterial, yeast, or insect cell by any method in the art. For example,the expression vector can be transferred into a host cell by physical,chemical, or biological means.

Physical methods for introducing a polynucleotide into a host cellinclude calcium phosphate precipitation, lipofection, particlebombardment, microinjection, electroporation, and the like. Methods forproducing cells comprising vectors and/or exogenous nucleic acids arewell-known in the art. See, for example, Sambrook et al., 2012,MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring HarborPress, NY). Nucleic acids can be introduced into target cells usingcommercially available methods which include electroporation (AmaxaNucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX)(Harvard Instruments, Boston, Mass.) or the Gene Pulser II (BioRad,Denver, Colo.), Multiporator (Eppendort, Hamburg Germany). Nucleic acidscan also be introduced into cells using cationic liposome mediatedtransfection using lipofection, using polymer encapsulation, usingpeptide mediated transfection, or using biolistic particle deliverysystems such as “gene guns” (see, for example, Nishikawa, et al. HumGene Ther., 12(8):861-70 (2001).

Biological methods for introducing a polynucleotide of interest into ahost cell include the use of DNA and RNA vectors. RNA vectors includevectors having a RNA promoter and/other relevant domains for productionof a RNA transcript. Viral vectors, and especially retroviral vectors,have become the most widely used method for inserting genes intomammalian, e.g., human cells. Other viral vectors may be derived fromlentivirus, poxviruses, herpes simplex virus, adenoviruses andadeno-associated viruses, and the like. See, for example, U.S. Pat. Nos.5,350,674 and 5,585,362.

Chemical means for introducing a polynucleotide into a host cell includecolloidal dispersion systems, such as macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes. Anexemplary colloidal system for use as a delivery vehicle in vitro and invivo is a liposome (e.g., an artificial membrane vesicle).

In the case where a non-viral delivery system is utilized, an exemplarydelivery vehicle is a liposome. The use of lipid formulations iscontemplated for the introduction of the nucleic acids into a host cell(in vitro, ex vivo or in vivo). In another aspect, the nucleic acid maybe associated with a lipid. The nucleic acid associated with a lipid maybe encapsulated in the aqueous interior of a liposome, interspersedwithin the lipid bilayer of a liposome, attached to a liposome via alinking molecule that is associated with both the liposome and theoligonucleotide, entrapped in a liposome, complexed with a liposome,dispersed in a solution containing a lipid, mixed with a lipid, combinedwith a lipid, contained as a suspension in a lipid, contained orcomplexed with a micelle, or otherwise associated with a lipid. Lipid,lipid/DNA or lipid/expression vector associated compositions are notlimited to any particular structure in solution. For example, they maybe present in a bilayer structure, as micelles, or with a “collapsed”structure. They may also simply be interspersed in a solution, possiblyforming aggregates that are not uniform in size or shape. Lipids arefatty substances which may be naturally occurring or synthetic lipids.For example, lipids include the fatty droplets that naturally occur inthe cytoplasm as well as the class of compounds which contain long-chainaliphatic hydrocarbons and their derivatives, such as fatty acids,alcohols, amines, amino alcohols, and aldehydes.

Lipids suitable for use can be obtained from commercial sources. Forexample, dimyristyl phosphatidylcholine (“DMPC”) can be obtained fromSigma, St. Louis, Mo.; dicetyl phosphate (“DCP”) can be obtained from K& K Laboratories (Plainview, N.Y.); cholesterol (“Choi”) can be obtainedfrom Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) andother lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham,Ala.). Stock solutions of lipids in chloroform or chloroform/methanolcan be stored at about −20° C. Chloroform is used as the only solventsince it is more readily evaporated than methanol. “Liposome” is ageneric term encompassing a variety of single and multilamellar lipidvehicles formed by the generation of enclosed lipid bilayers oraggregates. Liposomes can be characterized as having vesicularstructures with a phospholipid bilayer membrane and an inner aqueousmedium. Multilamellar liposomes have multiple lipid layers separated byaqueous medium. They form spontaneously when phospholipids are suspendedin an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh et al.,1991 Glycobiology 5: 505-10). However, compositions that have differentstructures in solution than the normal vesicular structure are alsoencompassed. For example, the lipids may assume a micellar structure ormerely exist as nonuniform aggregates of lipid molecules. Alsocontemplated are lipofectamine-nucleic acid complexes.

Regardless of the method used to introduce exogenous nucleic acids intoa host cell or otherwise expose a cell to the molecules describedherein, in order to confirm the presence of the nucleic acids in thehost cell, a variety of assays may be performed. Such assays include,for example, “molecular biological” assays well known to those of skillin the art, such as Southern and Northern blotting, RT-PCR and PCR;“biochemical” assays, such as detecting the presence or absence of aparticular peptide, e.g., by immunological means (ELISAs and Westernblots) or by assays described herein to identify agents falling withinthe scope of the invention.

In one embodiment, one or more of the nucleic acid sequences areintroduced by a method selected from the group consisting of transducingthe population of cells, transfecting the population of cells, andelectroporating the population of cells. In one embodiment, a populationof cells comprises one or more of the nucleic acid sequences describedherein.

In one embodiment, the nucleic acids introduced into the cell are RNA.In another embodiment, the RNA is mRNA that comprises in vitrotranscribed RNA or synthetic RNA. The RNA is produced by in vitrotranscription using a polymerase chain reaction (PCR)-generatedtemplate. DNA of interest from any source can be directly converted byPCR into a template for in vitro mRNA synthesis using appropriateprimers and RNA polymerase. The source of the DNA can be, for example,genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence or anyother appropriate source of DNA. The desired template for in vitrotranscription is a CAR.

PCR can be used to generate a template for in vitro transcription ofmRNA which is then introduced into cells. Methods for performing PCR arewell known in the art. Primers for use in PCR are designed to haveregions that are substantially complementary to regions of the DNA to beused as a template for the PCR. “Substantially complementary”, as usedherein, refers to sequences of nucleotides where a majority or all ofthe bases in the primer sequence are complementary, or one or more basesare non-complementary, or mismatched. Substantially complementarysequences are able to anneal or hybridize with the intended DNA targetunder annealing conditions used for PCR. The primers can be designed tobe substantially complementary to any portion of the DNA template. Forexample, the primers can be designed to amplify the portion of a genethat is normally transcribed in cells (the open reading frame),including 5′ and 3′ UTRs. The primers can also be designed to amplify aportion of a gene that encodes a particular domain of interest. In oneembodiment, the primers are designed to amplify the coding region of ahuman cDNA, including all or portions of the 5′ and 3′ UTRs. Primersuseful for PCR are generated by synthetic methods that are well known inthe art. “Forward primers” are primers that contain a region ofnucleotides that are substantially complementary to nucleotides on theDNA template that are upstream of the DNA sequence that is to beamplified. “Upstream” is used herein to refer to a location 5, to theDNA sequence to be amplified relative to the coding strand. “Reverseprimers” are primers that contain a region of nucleotides that aresubstantially complementary to a double-stranded DNA template that aredownstream of the DNA sequence that is to be amplified. “Downstream” isused herein to refer to a location 3′ to the DNA sequence to beamplified relative to the coding strand.

Chemical structures that have the ability to promote stability and/ortranslation efficiency of the RNA may also be used. The RNA preferablyhas 5′ and 3′ UTRs. In one embodiment, the 5′ UTR is between zero and3000 nucleotides in length. The length of 5′ and 3′ UTR sequences to beadded to the coding region can be altered by different methods,including, but not limited to, designing primers for PCR that anneal todifferent regions of the UTRs. Using this approach, one of ordinaryskill in the art can modify the 5′ and 3′ UTR lengths required toachieve optimal translation efficiency following transfection of thetranscribed RNA.

The 5′ and 3′ UTRs can be the naturally occurring, endogenous 5′ and 3′UTRs for the gene of interest. Alternatively, UTR sequences that are notendogenous to the gene of interest can be added by incorporating the UTRsequences into the forward and reverse primers or by any othermodifications of the template. The use of UTR sequences that are notendogenous to the gene of interest can be useful for modifying thestability and/or translation efficiency of the RNA. For example, it isknown that AU-rich elements in 3′ UTR sequences can decrease thestability of mRNA. Therefore, 3′ UTRs can be selected or designed toincrease the stability of the transcribed RNA based on properties ofUTRs that are well known in the art.

In one embodiment, the 5′ UTR can contain the Kozak sequence of theendogenous gene. Alternatively, when a 5′ UTR that is not endogenous tothe gene of interest is being added by PCR as described above, aconsensus Kozak sequence can be redesigned by adding the 5′ UTRsequence. Kozak sequences can increase the efficiency of translation ofsome RNA transcripts, but does not appear to be required for all RNAs toenable efficient translation. The requirement for Kozak sequences formany mRNAs is known in the art. In other embodiments the 5′ UTR can bederived from an RNA virus whose RNA genome is stable in cells. In otherembodiments various nucleotide analogues can be used in the 3′ or 5′ UTRto impede exonuclease degradation of the mRNA.

To enable synthesis of RNA from a DNA template without the need for genecloning, a promoter of transcription should be attached to the DNAtemplate upstream of the sequence to be transcribed. When a sequencethat functions as a promoter for an RNA polymerase is added to the 5′end of the forward primer, the RNA polymerase promoter becomesincorporated into the PCR product upstream of the open reading framethat is to be transcribed. In one embodiment, the promoter is a T7polymerase promoter, as described elsewhere herein. Other usefulpromoters include, but are not limited to, T3 and SP6 RNA polymerasepromoters. Consensus nucleotide sequences for T7, T3 and SP6 promotersare known in the art.

In one embodiment, the mRNA has both a cap on the 5′ end and a 3′poly(A) tail which determine ribosome binding, initiation of translationand stability mRNA in the cell. On a circular DNA template, forinstance, plasmid DNA, RNA polymerase produces a long concatamericproduct which is not suitable for expression in eukaryotic cells. Thetranscription of plasmid DNA linearized at the end of the 3′ UTR resultsin normal sized mRNA which is not effective in eukaryotic transfectioneven if it is polyadenylated after transcription.

On a linear DNA template, phage T7 RNA polymerase can extend the 3′ endof the transcript beyond the last base of the template (Schenborn andMierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva andBerzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003).

The conventional method of integration of polyA/T stretches into a DNAtemplate is molecular cloning. However polyA/T sequence integrated intoplasmid DNA can cause plasmid instability, which is why plasmid DNAtemplates obtained from bacterial cells are often highly contaminatedwith deletions and other aberrations. This makes cloning procedures notonly laborious and time consuming but often not reliable. That is why amethod which allows construction of DNA templates with polyA/T 3′stretch without cloning highly desirable.

The polyA/T segment of the transcriptional DNA template can be producedduring PCR by using a reverse primer containing a polyT tail, such as100T tail (size can be 50-5000 T), or after PCR by any other method,including, but not limited to, DNA ligation or in vitro recombination.Poly(A) tails also provide stability to RNAs and reduce theirdegradation. Generally, the length of a poly(A) tail positivelycorrelates with the stability of the transcribed RNA. In one embodiment,the poly(A) tail is between 100 and 5000 adenosines.

Poly(A) tails of RNAs can be further extended following in vitrotranscription with the use of a poly(A) polymerase, such as E. colipolyA polymerase (E-PAP). In one embodiment, increasing the length of apoly(A) tail from 100 nucleotides to between 300 and 400 nucleotidesresults in about a two-fold increase in the translation efficiency ofthe RNA. Additionally, the attachment of different chemical groups tothe 3′ end can increase mRNA stability. Such attachment can containmodified/artificial nucleotides, aptamers and other compounds. Forexample, ATP analogs can be incorporated into the poly(A) tail usingpoly(A) polymerase. ATP analogs can further increase the stability ofthe RNA.

5′ caps also provide stability to RNA molecules. In a preferredembodiment, RNAs produced by the methods disclosed herein include a 5′cap. The 5′ cap is provided using techniques known in the art anddescribed herein (Cougot, et al., Trends in Biochem. Sci., 29:436-444(2001); Stepinski, et al., RNA, 7:1468-95 (2001); Elango, et al.,Biochim. Biophys. Res. Commun., 330:958-966 (2005)).

The RNAs produced by the methods disclosed herein can also contain aninternal ribosome entry site (IRES) sequence. The IRES sequence may beany viral, chromosomal or artificially designed sequence which initiatescap-independent ribosome binding to mRNA and facilitates the initiationof translation. Any solutes suitable for cell electroporation, which cancontain factors facilitating cellular permeability and viability such assugars, peptides, lipids, proteins, antioxidants, and surfactants can beincluded.

Some in vitro-transcribed RNA (IVT-RNA) vectors are known in theliterature which are utilized in a standardized manner as template forin vitro transcription and which have been genetically modified in sucha way that stabilized RNA transcripts are produced. Currently protocolsused in the art are based on a plasmid vector with the followingstructure: a 5′ RNA polymerase promoter enabling RNA transcription,followed by a gene of interest which is flanked either 3′ and/or 5′ byuntranslated regions (UTR), and a 3′ polyadenyl cassette containing50-70 A nucleotides. Prior to in vitro transcription, the circularplasmid is linearized downstream of the polyadenyl cassette by type IIrestriction enzymes (recognition sequence corresponds to cleavage site).The polyadenyl cassette thus corresponds to the later poly(A) sequencein the transcript. As a result of this procedure, some nucleotidesremain as part of the enzyme cleavage site after linearization andextend or mask the poly(A) sequence at the 3′ end. It is not clear,whether this nonphysiological overhang affects the amount of proteinproduced intracellularly from such a construct.

In one aspect, the RNA construct is delivered into the cells byelectroporation. See, e.g., the formulations and methodology ofelectroporation of nucleic acid constructs into mammalian cells astaught in US 2004/0014645, US 2005/0052630A1, US 2005/0070841A1, US2004/0059285A1, US 2004/0092907A1. The various parameters includingelectric field strength required for electroporation of any known celltype are generally known in the relevant research literature as well asnumerous patents and applications in the field. See e.g., U.S. Pat. Nos.6,678,556, 7,171,264, and 7,173,116. Apparatus for therapeuticapplication of electroporation are available commercially, e.g., theMedPulser™ DNA Electroporation Therapy System (Inovio/Genetronics, SanDiego, Calif.), and are described in patents such as U.S. Pat. Nos.6,567,694; 6,516,223, 5,993,434, 6,181,964, 6,241,701, and 6,233,482;electroporation may also be used for transfection of cells in vitro asdescribed e.g. in US20070128708A1. Electroporation may also be utilizedto deliver nucleic acids into cells in vitro. Accordingly,electroporation-mediated administration into cells of nucleic acidsincluding expression constructs utilizing any of the many availabledevices and electroporation systems known to those of skill in the artpresents an exciting new means for delivering an RNA of interest to atarget cell.

Sources of Cells

In one embodiment, phagocytic cells are used in the compositions andmethods described herein. A source of phagocytic cells, such asmonocytes, macrophages and/or dendritic cells, is obtained from asubject. Non-limiting examples of subjects include humans, dogs, cats,mice, rats, and transgenic species thereof. Preferably, the subject is ahuman. The cells can be obtained from a number of sources, includingperipheral blood mononuclear cells, bone marrow, lymph node tissue,spleen tissue, umbilical cord, and tumors. In certain embodiments, anynumber of monocyte, macrophage, dendritic cell or progenitor cell linesavailable in the art, may be used. In certain embodiments, the cells canbe obtained from a unit of blood collected from a subject using anynumber of techniques known to the skilled artisan, such as Ficollseparation. In one embodiment, cells from the circulating blood of anindividual are obtained by apheresis or leukapheresis. The apheresisproduct typically contains lymphocytes, including T cells, monocytes,granulocytes, B cells, other nucleated white blood cells, red bloodcells, and platelets. The cells collected by apheresis may be washed toremove the plasma fraction and to place the cells in an appropriatebuffer or media, such as phosphate buffered saline (PBS) or washsolution lacks calcium and may lack magnesium or may lack many if notall divalent cations, for subsequent processing steps. After washing,the cells may be resuspended in a variety of biocompatible buffers, suchas, for example, Ca-free, Mg-free PBS. Alternatively, the undesirablecomponents of the apheresis sample may be removed and the cells directlyresuspended in culture media.

In another embodiment, cells are isolated from peripheral blood bylysing the red blood cells and depleting the lymphocytes and red bloodcells, for example, by centrifugation through a PERCOLL™ gradient.Alternatively, cells can be isolated from umbilical cord. In any event,a specific subpopulation of the monocytes, macrophages and/or dendriticcells can be further isolated by positive or negative selectiontechniques.

The mononuclear cells so isolated can be depleted of cells expressingcertain antigens, including, but not limited to, CD34, CD3, CD4, CD8,CD14, CD19 or CD20. Depletion of these cells can be accomplished usingan isolated antibody, a biological sample comprising an antibody, suchas ascites fluid, an antibody bound to a physical support, and a cellbound antibody.

Enrichment of a monocyte, macrophage and/or dendritic cell population bynegative selection can be accomplished using a combination of antibodiesdirected to surface markers unique to the negatively selected cells. Apreferred method is cell sorting and/or selection via negative magneticimmunoadherence or flow cytometry that uses a cocktail of monoclonalantibodies directed to cell surface markers present on the cellsnegatively selected. For example, enrich of a cell population formonocytes, macrophages and/or dendritic cells by negative selection canbe accomplished using a monoclonal antibody cocktail that typicallyincludes antibodies to CD34, CD3, CD4, CD8, CD14, CD19 or CD20.

During isolation of a desired population of cells by positive ornegative selection, the concentration of cells and surface (e.g.,particles such as beads) can be varied. In certain embodiments, it maybe desirable to significantly decrease the volume in which beads andcells are mixed together (i.e., increase the concentration of cells), toensure maximum contact of cells and beads. For example, in oneembodiment, a concentration of 2 billion cells/ml is used. In oneembodiment, a concentration of 1 billion cells/ml is used. In a furtherembodiment, greater than 100 million cells/ml is used. In a furtherembodiment, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45,or 50 million cells/ml is used. In yet another embodiment, aconcentration of cells from 75, 80, 85, 90, 95, or 100 million cells/mlis used. In further embodiments, concentrations of 125 or 150 millioncells/ml can be used. The use of high concentrations of cells can resultin increased cell yield, cell activation, and cell expansion.

In one embodiment, a population of cells comprises the monocytes,macrophages, or dendritic cells of the present invention. Examples of apopulation of cells include, but are not limited to, peripheral bloodmononuclear cells, cord blood cells, a purified population of monocytes,macrophages, or dendritic cells, and a cell line. In another embodiment,peripheral blood mononuclear cells comprise the population of monocytes,macrophages, or dendritic cells. In yet another embodiment, purifiedcells comprise the population of monocytes, macrophages, or dendriticcells.

In another embodiment, the cells have upregulated M1 markers anddownregulated M2 markers. For example, at least one M1 marker, such asHLA DR, CD86, CD80, and PDL1, is upregulated in the phagocytic cell. Inanother example, at least one M2 marker, such as CD206, CD163, isdownregulated in the phagocytic cell. In one embodiment, the cell has atleast one upregulated M1 marker and at least one downregulated M2marker.

In yet another embodiment, targeted effector activity in the phagocyticcell is enhanced by inhibition of either CD47 or SIRPα activity. CD47and/or SIRPα activity may be inhibited by treating the phagocytic cellwith an anti-CD47 or anti-SIRPα antibody. Alternatively, CD47 or SIRPαactivity may be inhibited by any method known to those skilled in theart.

Expansion of Cells

In one embodiment, the cells or population of cells comprisingmonocytes, macrophages, or dendritic cells are cultured for expansion.In another embodiment, the cells or population of cells comprisingprogenitor cells are cultured for differentiation and expansion ofmonocytes, macrophages, or dendritic cells. The present inventioncomprises expanding a population of monocytes, macrophages, or dendriticcells comprising a chimeric antigen receptor as described herein.

As demonstrated by the data disclosed herein, expanding the cells by themethods disclosed herein can be multiplied by about 10 fold, 20 fold, 30fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, 100 fold,200 fold, 300 fold, 400 fold, 500 fold, 600 fold, 700 fold, 800 fold,900 fold, 1000 fold, 2000 fold, 3000 fold, 4000 fold, 5000 fold, 6000fold, 7000 fold, 8000 fold, 9000 fold, 10,000 fold, 100,000 fold,1,000,000 fold, 10,000,000 fold, or greater, and any and all whole orpartial integers therebetween. In one embodiment, the cells expand inthe range of about 20 fold to about 50 fold.

Following culturing, the cells can be incubated in cell medium in aculture apparatus for a period of time or until the cells reachconfluency or high cell density for optimal passage before passing thecells to another culture apparatus. The culturing apparatus can be ofany culture apparatus commonly used for culturing cells in vitro.Preferably, the level of confluence is 70% or greater before passing thecells to another culture apparatus. More preferably, the level ofconfluence is 90% or greater. A period of time can be any time suitablefor the culture of cells in vitro. The culture medium may be replacedduring the culture of the cells at any time. Preferably, the culturemedium is replaced about every 2 to 3 days. The cells are then harvestedfrom the culture apparatus whereupon the cells can be used immediatelyor stored for use at a later time

The culturing step as described herein (contact with agents as describedherein) can be very short, for example less than 24 hours such as 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,or 23 hours. The culturing step as described further herein (contactwith agents as described herein) can be longer, for example 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more days.

In one embodiment, the cells may be cultured for several hours (about 3hours) to about 14 days or any hourly integer value in between.Conditions appropriate for cell culture include an appropriate media(e.g., macrophage complete medium, DMEM/F12, DMEM/F12-10 (Invitrogen))that may contain factors necessary for proliferation and viability,including serum (e.g., fetal bovine or human serum), L-glutamine,insulin, M-CSF, GM-CSF, IL-10, IL-12, IL-15, TGF-beta, and TNF-α. or anyother additives for the growth of cells known to the skilled artisan.Other additives for the growth of cells include, but are not limited to,surfactant, plasmanate, and reducing agents such as N-acetyl-cysteineand 2-mercaptoethanol. Media can include RPMI 1640, AIM-V, DMEM, MEM,α-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, with added aminoacids, sodium pyruvate, and vitamins, either serum-free or supplementedwith an appropriate amount of serum (or plasma) or a defined set ofhormones, and/or an amount of cytokine(s) sufficient for the growth andexpansion of the cells. Antibiotics, e.g., penicillin and streptomycin,are included only in experimental cultures, not in cultures of cellsthat are to be infused into a subject. The target cells are maintainedunder conditions necessary to support growth, for example, anappropriate temperature (e.g., 37° C.) and atmosphere (e.g., air plus 5%CO2).

The medium used to culture the cells may include an agent that canactivate the cells. For example, an agent that is known in the art toactivate the monocyte, macrophage or dendritic cell is included in theculture medium.

Therapy

The modified cells described herein may be included in a composition fortreatment of a subject. In one aspect, the composition comprises themodified cell comprising the chimeric antigen receptor described herein.The composition may include a pharmaceutical composition and furtherinclude a pharmaceutically acceptable carrier. A therapeuticallyeffective amount of the pharmaceutical composition comprising themodified cells may be administered.

In one aspect, the invention includes a method of treating a disease orcondition associated with a tumor or cancer in a subject comprisingadministering to the subject a therapeutically effective amount of apharmaceutical composition comprising the modified cell describedherein. In another aspect, the invention includes a method of treating asolid tumor in a subject, comprising administering to the subject atherapeutically effective amount of a pharmaceutical compositioncomprising the modified cell described herein. In another aspect, theinvention includes a method for stimulating an immune response to atarget tumor cell or tumor tissue in a subject comprising administeringto a subject a therapeutically effective amount of a pharmaceuticalcomposition comprising the modified cell described herein. In yetanother aspect, the invention includes use of the modified celldescribed herein in the manufacture of a medicament for the treatment ofan immune response in a subject in need thereof. In still anotheraspect, the invention includes use of the modified cell described hereinin the manufacture of a medicament for the treatment of a tumor orcancer in a subject in need thereof.

The modified cells generated as described herein possess targetedeffector activity. In one embodiment, the modified cells have targetedeffector activity directed against an antigen on a target cell, such asthrough specific binding to an antigen binding domain of a CAR. Inanother embodiment, the targeted effector activity includes, but is notlimited to, phagocytosis, targeted cellular cytotoxicity, antigenpresentation, and cytokine secretion.

In another embodiment, the modified cell described herein has thecapacity to deliver an agent, a biological agent or a therapeutic agentto the target. The cell may be modified or engineered to deliver anagent to a target, wherein the agent is selected from the groupconsisting of a nucleic acid, an antibiotic, an anti-inflammatory agent,an antibody or antibody fragments thereof, a growth factor, a cytokine,an enzyme, a protein, a peptide, a fusion protein, a synthetic molecule,an organic molecule, a carbohydrate or the like, a lipid, a hormone, amicrosome, a derivative or a variation thereof, and any combinationthereof. As a non-limiting example, a macrophage modified with a CARthat targets a tumor antigen is capable of secreting an agent, such as acytokine or antibody, to aid in macrophage function. Antibodies, such asanti-CD47/antiSIRPα mAB, may also aid in macrophage function. In yetanother example, the macrophage modified with a CAR that targets a tumorantigen is engineered to encode a siRNA that aids macrophage function bydownregulating inhibitory genes (i.e. SIRPα). Another example, the CARmacrophage is engineered to express a dominant negative (or otherwisemutated) version of a receptor or enzyme that aids in macrophagefunction.

In one embodiment, the macrophage is modified with multiple genes,wherein at least one gene includes a CAR and at least one other genecomprises a genetic element that enhances CAR macrophage function. Inanother embodiment, the macrophage is modified with multiple genes,wherein at least one gene includes a CAR and at least one other geneaids or reprograms the function of other immune cells (such as T cellswithin the tumor microenvironment).

Further, the modified cells can be administered to an animal, preferablya mammal, even more preferably a human, to suppress an immune reaction,such as those common to autoimmune diseases such as diabetes, psoriasis,rheumatoid arthritis, multiple sclerosis, GVHD, enhancing allografttolerance induction, transplant rejection, and the like. In addition,the cells of the present invention can be used for the treatment of anycondition in which a diminished or otherwise inhibited immune response,especially a cell-mediated immune response, is desirable to treat oralleviate the disease. In one aspect, the invention includes treating acondition, such as an autoimmune disease, in a subject, comprisingadministering to the subject a therapeutically effective amount of apharmaceutical composition comprising a population of the cellsdescribed herein. In addition, the cells of the present invention can beadministered as pre-treatment or conditioning prior to treatment with analternative anti-cancer immunotherapy, including but not limited to CART cells, tumor-infiltrating lymphocyte, or a checkpoint inhibitor.

Examples of autoimmune disease include but are not limited to, AcquiredImmunodeficiency Syndrome (AIDS, which is a viral disease with anautoimmune component), alopecia areata, ankylosing spondylitis,antiphospholipid syndrome, autoimmune Addison's disease, autoimmunehemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease(AIED), autoimmune lymphoproliferative syndrome (ALPS), autoimmunethrombocytopenic purpura (ATP), Behcet's disease, cardiomyopathy, celiacsprue-dermatitis hepetiformis; chronic fatigue immune dysfunctionsyndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy(CIPD), cicatricial pemphigold, cold agglutinin disease, crest syndrome,Crohn's disease, Degos' disease, dermatomyositis-juvenile, discoidlupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis,Graves' disease, Guillain-Barre syndrome, Hashimoto's thyroiditis,idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura(ITP), IgA nephropathy, insulin-dependent diabetes mellitus, juvenilechronic arthritis (Still's disease), juvenile rheumatoid arthritis,Meniere's disease, mixed connective tissue disease, multiple sclerosis,myasthenia gravis, pernacious anemia, polyarteritis nodosa,polychondritis, polyglandular syndromes, polymyalgia rheumatica,polymyositis and dermatomyositis, primary agammaglobulinemia, primarybiliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomena,Reiter's syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis,scleroderma (progressive systemic sclerosis (PSS), also known assystemic sclerosis (SS)), Sjogren's syndrome, stiff-man syndrome,systemic lupus erythematosus, Takayasu arteritis, temporalarteritis/giant cell arteritis, ulcerative colitis, uveitis, vitiligoand Wegener's granulomatosis.

The cells can also be used to treat inflammatory disorders. Examples ofinflammatory disorders include but are not limited to, chronic and acuteinflammatory disorders. Examples of inflammatory disorders includeAlzheimer's disease, asthma, atopic allergy, allergy, atherosclerosis,bronchial asthma, eczema, glomerulonephritis, graft vs. host disease,hemolytic anemias, osteoarthritis, sepsis, stroke, transplantation oftissue and organs, vasculitis, diabetic retinopathy and ventilatorinduced lung injury.

The cells of the present invention can be used to treat cancers. Cancersinclude tumors that are not vascularized, or not yet substantiallyvascularized, as well as vascularized tumors. The cancers may comprisenon-solid tumors (such as hematological tumors, for example, leukemiasand lymphomas) or may comprise solid tumors. Types of cancers to betreated with the cells of the invention include, but are not limited to,carcinoma, blastoma, and sarcoma, and certain leukemia or lymphoidmalignancies, benign and malignant tumors, and malignancies e.g.,sarcomas, carcinomas, and melanomas. Adult tumors/cancers and pediatrictumors/cancers are also included.

Solid tumors are abnormal masses of tissue that usually do not containcysts or liquid areas. Solid tumors can be benign or malignant.Different types of solid tumors are named for the type of cells thatform them (such as sarcomas, carcinomas, and lymphomas). Examples ofsolid tumors, such as sarcomas and carcinomas, include fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and othersarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreaticcancer, breast cancer, lung cancers, ovarian cancer, prostate cancer,hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma,adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma,papillary thyroid carcinoma, pheochromocytomas sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas, medullarycarcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bileduct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer,testicular tumor, seminoma, bladder carcinoma, melanoma, and CNS tumors(such as a glioma (such as brainstem glioma and mixed gliomas),glioblastoma (also known as glioblastoma multiforme) astrocytoma, CNSlymphoma, germinoma, medulloblastoma, Schwannoma craniopharyogioma,ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,oligodendroglioma, menangioma, neuroblastoma, retinoblastoma and brainmetastases).

Hematologic cancers are cancers of the blood or bone marrow. Examples ofhematological (or hematogenous) cancers include leukemias, includingacute leukemias (such as acute lymphocytic leukemia, acute myelocyticleukemia, acute myelogenous leukemia and myeloblastic, promyelocytic,myelomonocytic, monocytic and erythroleukemia), chronic leukemias (suchas chronic myelocytic (granulocytic) leukemia, chronic myelogenousleukemia, and chronic lymphocytic leukemia), polycythemia vera,lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and highgrade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavychain disease, myelodysplastic syndrome, hairy cell leukemia andmyelodysplasia.

Cells of the invention can be administered in dosages and routes and attimes to be determined in appropriate pre-clinical and clinicalexperimentation and trials. Cell compositions may be administeredmultiple times at dosages within these ranges. Administration of hecells of the invention may be combined with other methods useful totreat the desired disease or condition as determined by those of skillin the art.

The cells of the invention to be administered may be autologous,allogeneic or xenogeneic with respect to the subject undergoing therapy.

The administration of the cells of the invention may be carried out inany convenient manner known to those of skill in the art. The cells ofthe present invention may be administered to a subject by aerosolinhalation, injection, ingestion, transfusion, implantation ortransplantation. The compositions described herein may be administeredto a patient transarterially, subcutaneously, intradermally,intratumorally, intranodally, intramedullary, intramuscularly, byintravenous (i.v.) injection, or intraperitoneally. In other instances,the cells of the invention are injected directly into a site ofinflammation in the subject, a local disease site in the subject, alymphnode, an organ, a tumor, and the like.

Pharmaceutical Compositions

Pharmaceutical compositions of the present invention may comprise thecells as described herein, in combination with one or morepharmaceutically or physiologically acceptable carriers, diluents orexcipients. Such compositions may comprise buffers such as neutralbuffered saline, phosphate buffered saline and the like; carbohydratessuch as glucose, mannose, sucrose or dextrans, mannitol; proteins;polypeptides or amino acids such as glycine; antioxidants; chelatingagents such as EDTA or glutathione; adjuvants (e.g., aluminumhydroxide); and preservatives. Compositions of the present invention arepreferably formulated for intravenous administration.

Pharmaceutical compositions of the present invention may be administeredin a manner appropriate to the disease to be treated (or prevented). Thequantity and frequency of administration will be determined by suchfactors as the condition of the patient, and the type and severity ofthe patient's disease, although appropriate dosages may be determined byclinical trials.

When “an immunologically effective amount”, “an anti-immune responseeffective amount”, “an immune response-inhibiting effective amount”, or“therapeutic amount” is indicated, the precise amount of thecompositions of the present invention to be administered can bedetermined by a physician with consideration of individual differencesin age, weight, immune response, and condition of the patient (subject).It can generally be stated that a pharmaceutical composition comprisingthe cells described herein may be administered at a dosage of 10⁴ to 10⁹cell s/kg body weight, preferably 10⁵ to 10⁶ cells/kg body weight,including all integer values within those ranges. The cell compositionsdescribed herein may also be administered multiple times at thesedosages. The cells can be administered by using infusion techniques thatare commonly known in immunotherapy (see, e.g., Rosenberg et al., NewEng. J. of Med. 319:1676, 1988). The optimal dosage and treatment regimefor a particular patient can readily be determined by one skilled in theart of medicine by monitoring the patient for signs of disease andadjusting the treatment accordingly.

In certain embodiments, it may be desired to administer monocytes,macrophages, or dendritic cells to a subject and then subsequentlyredraw blood (or have an apheresis performed), activate the monocytes,macrophages, or dendritic cells therefrom according to the presentinvention, and reinfuse the patient with these activated cells. Thisprocess can be carried out multiple times every few weeks. In certainembodiments, the cells can be activated from blood draws of from 10 mlto 400 ml. In certain embodiments, the cells are activated from blooddraws of 20 ml, 30 ml, 40 ml, 50 ml, 60 ml, 70 ml, 80 ml, 90 ml, or 100ml. Not to be bound by theory, using this multiple blood draw/multiplereinfusion protocol, may select out certain populations of cells.

In certain embodiments of the present invention, cells are modifiedusing the methods described herein, or other methods known in the artwhere the cells are expanded to therapeutic levels, are administered toa patient in conjunction with (e.g., before, simultaneously orfollowing) any number of relevant treatment modalities, including butnot limited to treatment with agents such as antiviral therapy,cidofovir and interleukin-2, Cytarabine (also known as ARA-C) ornatalizumab treatment for MS patients or treatments for PML patients. Infurther embodiments, the cells of the invention may be used incombination with CART cell therapy, chemotherapy, radiation,immunosuppressive agents, such as cyclosporin, azathioprine,methotrexate, mycophenolate, and FK506, antibodies, or otherimmunoablative agents such as anti-CD52 antibody alemtuzumab (CAM PATH),anti-CD3 antibodies or other antibody therapies, cytoxin, fludaribine,cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228,cytokines, and irradiation. These drugs inhibit either the calciumdependent phosphatase calcineurin (cyclosporine and FK506) or inhibitthe p70S6 kinase that is important for growth factor induced signaling(rapamycin). (Liu et al., Cell 66:807-815, 1991; Henderson et al.,Immun. 73:316-321, 1991; Bierer et al., Curr. Opin. Immun. 5:763-773,1993). In a further embodiment, the cell compositions of the presentinvention are administered to a patient in conjunction with (e.g.,before, simultaneously or following) bone marrow transplantation,lymphocyte ablative therapy using either chemotherapy agents such as,fludarabine, external-beam radiation therapy (XRT), cyclophosphamide,Rituxan, or antibodies such as OKT3 or CAMPATH. For example, in oneembodiment, subjects may undergo standard treatment with high dosechemotherapy followed by peripheral blood stem cell transplantation. Incertain embodiments, following the transplant, subjects receive aninfusion of the cells of the present invention. In an additionalembodiment, the cells may be administered before or following surgery.

The dosage of the above treatments to be administered to a subject willvary with the precise nature of the condition being treated and therecipient of the treatment. The scaling of dosages for humanadministration can be performed according to art-accepted practices. Thedose for CAMPATH, for example, will generally be in the range 1 to about100 mg for an adult patient, usually administered daily for a periodbetween 1 and 30 days. The preferred daily dose is 1 to 10 mg per dayalthough in some instances larger doses of up to 40 mg per day may beused (described in U.S. Pat. No. 6,120,766).

It should be understood that the method and compositions that would beuseful in the present invention are not limited to the particularformulations set forth in the examples. The following examples are putforth so as to provide those of ordinary skill in the art with acomplete disclosure and description of how to make and use the cells,expansion and culture methods, and therapeutic methods of the invention,and are not intended to limit the scope of what the inventors regard astheir invention.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are well within the purview of the skilled artisan.Such techniques are explained fully in the literature, such as,“Molecular Cloning: A Laboratory Manual”, fourth edition (Sambrook,2012); “Oligonucleotide Synthesis” (Gait, 1984); “Culture of AnimalCells” (Freshney, 2010); “Methods in Enzymology” “Handbook ofExperimental Immunology” (Weir, 1997); “Gene Transfer Vectors forMammalian Cells” (Miller and Calos, 1987); “Short Protocols in MolecularBiology” (Ausubel, 2002); “Polymerase Chain Reaction: Principles,Applications and Troubleshooting”, (Babar, 2011); “Current Protocols inImmunology” (Coligan, 2002). These techniques are applicable to theproduction of the polynucleotides and polypeptides of the invention,and, as such, may be considered in making and practicing the invention.Particularly useful techniques for particular embodiments will bediscussed in the sections that follow.

EXPERIMENTAL EXAMPLES

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the invention should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the compounds of the presentinvention and practice the claimed methods. The following workingexamples therefore, specifically point out the preferred embodiments ofthe present invention, and are not to be construed as limiting in anyway the remainder of the disclosure.

The materials and methods employed in these experiments are nowdescribed.

Cell Culture: THP1, K562, SKOV3, SKBR3, HDLM2, MD468, and all cell lineswere cultured in RPMI 1640 supplemented with 10% fetal bovine serum andpenicillin/streptomycin at 37C in 5% CO2. A THP1 mRFP+ subline (Wt) wasgenerated by lentiviral transduction and FACS purification of mRFP+ celllines. The THP1 mRFP+ subline was used to generate THP1 mRFP+CAR19z+(CAR19z; CARMA19z), THP1 mRFP+ CAR19Δz+(CAR19Δz; CARMA19Δz), THP1mRFP+ MesoZ+ and THP1 mRFP+ CARHer2z+(CARHer2z; CARMAHer2z) sublines.Monocyte differentiation was induced by culturing cells for 48 hourswith 1 ng/mL phorbol 12-myristate 13-acetate in culture media.

Primary Human Macrophages: Primary human monocytes were purified fromnormal donor apheresis product using Miltenyi CD14 MicroBeads (Miltenyi,130-050-201). Monocytes were cultured in X-Vivo media supplemented with5% human AB serum or RPMI 1640 supplemented with 10% fetal bovine serum,with penicillin/streptomycin, glutamax, and 10 ng/mL recombinant humanGM-CSF (PeproTech, 300-03) for 7 days in MACS GMP Cell DifferentiationBags (Miltenyi, 170-076-400). Macrophages were harvested on day 7 andcryopreserved in FBS+10% DMSO pending subsequent use.

Phagocytosis Assay: Wt or CARMA mRFP+THP1 sublines were differentiatedfor 48 hours with 1 ng/mL phorbol 12-myristate 13-acetate. GFP+antigenbearing tumor sublines, i.e. K562 CD19+ GFP+ cells, were added to thedifferentiated THP1 macrophages at a 1:1 ratio following PMA washout.Macrophages were co-cultured with target tumor cells for 4 hours, andphagocytosis was quantified by fluorescent microscopy using the EVOS FLAuto Cell Imaging System. An average of three fields of view wasconsidered as n, and all conditions were quantified in triplicates. FACSbased phagocytosis was analyzed on a BD LSR-Fortessa. FlowJo (Treestar,Inc.) was used to analyze flow cytometric data. Live, singlets gatedmRFP/GFP double positive events were considered phagocytosis. CD47/SIRPαaxis blockade was performed via addition of blocking monoclonalantibodies at the initiation of co-culture at indicated concentrations(mouse anti-human CD47 clone B6H12, eBioscience #14-0479-82; mouseanti-human CD47 clone 2D3 as negative control, eBioscience #14-0478-82;mouse anti-human SIRPα clone SE5A5, BioLegend #323802). TLRco-stimulation was performed by adding TLR1-9 agonists (Human TLR 1-9agonist kit; Invivogen #tlrl-kit1hw) at the time of co-culture.

In Vitro Killing Assay: Wt or CAR bearing macrophages were co-culturedwith antigen-bearing or control click-beetle green luciferase(CBG)/green fluorescent protein (GFP) positive target tumor cells atvarying effector to target ratios (starting at 30:1 and decreasing inthree-fold dilutors). Bioluminescent imaging was utilized to determinetumor burden, using the IVIS Spectrum Imaging System (Perkin Elmer).Percent specific lysis was calculated as follows:

% Specific Lysis=((Treated well−Tumor alone well)/(Maximal killing−tumoralone well)*100)

Time-Lapse Microscopy: Fluorescent time-lapse video microscopy of CARmediated phagocytosis was performed using the EVOS FL Auto Cell ImagingSystem. Images were captured every 40 seconds for 18 hours. Imageanalysis was performed with FIJI imaging software.

Lentiviral production and transfection: Chimeric antigen receptorconstructs were de novo synthesized by GeneArt (Life Technologies) andcloned into a lentiviral vector as previously described. Concentratedlentivirus was generated using HEK293T cells as previously described.

Adenoviral production and transfection: Ad5f35 chimeric adenoviralvectors encoding GFP, CAR, or no transgene under a CMV promoter wereproduced and titrated as per standard molecular biology procedure.Primary human macrophages were transduced with varying multiplicities ofinfection and serially imaged for GFP expression and viability using theEVOS FL Auto Cell Imaging System. CAR expression was assessed by FACSanalysis of surface CAR expression using His-tagged antigen andanti-His-APC secondary antibody (R&D Biosystems Clone AD1.1.10).

Flow Cytometry: FACS was performed on a BD LSR Fortessa. Surface CARexpression was detected with biotinylated protein L (GenScript M00097)and streptavidin APC (BioLegend, #405207) or His-tagged antigen andanti-His-APC secondary antibody (R&D Biosystems Clone AD1.1.10). Fcreceptors were blocked with Human Trustain FcX (BioLegend, #422301)prior to staining. CD47 expression was determined using mouse anti-humanCD47 APC (eBioscience #17-0479-41) with mouse IgG1 kappa APC isotypecontrol for background determination. Calreticulin expression wasdetermined with mouse anti-calreticulin PE clone FMC75 (Abcam #ab83220).All flow results are gated on live (Live/Dead Aqua Fixable Dead CellStain, Life Technologies L34957) single cells.

Imagestream Cytometry: FACS with single cell fluorescent imaging wasperformed on an ImageStream Mark II Imaging Flow Cytometer (EMDMillipore). Briefly, mRFP+ or DiI stained macrophages (CAR or control)were co-cultured with GFP+ tumor cells for 4 hours, prior to fixationand ImageStream data acquisition. Data was analyzed using ImageStreamsoftware (EMD Millipore).

RNA Electroporation: CAR constructs were cloned into in vitrotranscription plasmids under the control of a T7 promoter using standardmolecular biology techniques. CAR mRNA was in vitro transcribed using anmMessage mMachine T7 Ultra In Vitro Transcription Kit (Thermo Fisher),purified using RNEasy RNA Purification Kit (Qiagen), and electroporatedinto human macrophages using a BTX ECM850 electroporator (BTX HarvardApparatus). CAR expression was assayed at varying time pointspost-electroporation using FACS analysis.

TLR/Dectin-1 Priming: TLR or Dectin-1 priming in Wt or CAR macrophagesprior to in vitro phagocytosis or killing assays was performed bypre-incubating the cells with recommended doses of either TLR 1-9agonists (Human TLR1-9 Agonist Kit, Invivogen) or beta-glucan (MPBiomedicals, LLC), respectively, for 30 minutes prior to co-culture. Invitro function of Wt or CAR macrophages was compared between unprimedand primed conditions.

Macrophage/Monocyte Phenotype: The following surface markers wereassessed as part of a macrophage/monocyte immunophenotype FACS panel,for M1/M2 distinction: CD80, CD86, CD163, CD206, CD11B, HLA-DR,HLA-AB/C, PDL1, and PDL2 (BioLegend). TruStain FcX was used for Fcreceptor blockade prior to immunostaining. Macrophages/monocytes wereexposed to activating conditions, i.e. Ad5f35 transduction for 48 hours,or not, prior to phenotype assessment.

Seahorse Assay: Metabolic phenotype and oxygen consumption ofmacrophages was determined using the Seahorse assay (Seahorse XF,Agilent). Control or CAR macrophages were exposed to media control orimmunosuppressive cytokines for 24 hours prior to analysis. Cells weretreated with oligomycin, FCCP, and rotenone sequentially throughout theSeahorse assay. The assay was performed with 6 replicates per condition.

In Vivo Assays: NOD-scid IL2Rg-null-IL3/GM/SF, NSG-SGM3 (NSGS) mice wereused for human xenograft models. Mice engrafted with CBG-luciferasepositive human SKOV3 ovarian cancer cells were either left untreated, ortreated with untransduced, empty Ad5f35 transduced, or Ad5f35 CAR-HER2transduced human macrophages at different doses. Serial bioluminescentimaging was performed to monitor tumor burden (IVIS Spectrum, PerkinElmer). Organs and tumor were harvested upon sacrifice for FACSanalysis. Overall survival was monitored and compared using Kaplan-Meieranalysis.

The results of the experiments are now described.

FIG. 1A is a conceptual diagram of a chimeric antigen receptor (CAR)comprised of a gene/gene-product containing an extracellular domain withtargeting function, a hinge domain, a transmembrane domain, anintracellular signaling domain(s), and/or a 2A (P2A, T2A) forstoichiometric co-expression of an additional gene product which may ormay not be secreted, including any gene/transcript/protein, includingbut not limited to a cytokine, monoclonal antibody, antibody fragment,single chain variable fragment, enzyme, additional receptor, dominantnegative receptor, tumor associated antigen(s), and any combinationthereof. In addition, the CAR construct may include co-delivery ofCRISPR/Cas9 gene editing material, or be introduced in the context of aCRISPR/Cas9 pre-edited cell. Specific examples of CAR constructs aremodeled in FIG. 1B, including CARMA-ζ, CARMA-γ, and CARMA-Dectin, whichcontain an antigen specific scFv, CD8 hinge, CD8 transmembrane, and aCD3 ζ, FcεRI common γ subunit, or the intracellular domain of Dectin-1,respectively.

FIG. 2A is a graph showing CAR19z expressed on the surface of myeloidcells post lentiviral transduction. CAR19z lentivirus was titrated inthree-fold dilutors and used to transduce 1e5/0.1 mL mRFP+THP1 cells.mRFP is a reporter gene (red fluorescent protein) that was expressed bylentiviral transduction of the myeloid cell line THP1. These cells canbe induced to differentiate to macrophages upon exposure to the chemicalPMA. THP1 cells were harvested 24 hours post-transduction and stainedfor CAR surface expression with biotinylated-protein L followed bystreptavidin-APC. Transduced THP1 cells were expanded and sorted by FACSto generate a 100% CAR19z positive mRFP+THP1 subline (FIG. 2B). FIG. 2Cdemonstrates expression of anti-CD19, anti-HER2, and anti-mesothelinlentiviral CAR constructs on THP1 macrophages, with CAR(+) events in theupper right quadrant.

FIG. 3A is a flow chart showing the overview of CARMA subline generationusing a THP1 macrophage model, differentiation with 1 ng/mL phorbol12-myristate 13-acetate (PMA), and in vitro phagocytosis assay.Anti-CD19, anti-HER2, and anti-mesothelin CAR macrophages, but not wildtype (Wt) macrophages, phagocytosed K562 tumor cells that expressedCD19, HER2, or mesothelin, respectively, as demonstrated by fluorescentmicroscopy based phagocytosis assays (FIGS. 3B-3D). CARMA tumorphagocytosis was further validated by a flow cytometric based assay, inwhich mRFP+CARMA against CD19 were co-cultured with CD19+ GFP+K562 cellsand double positive events were quantified (representative FACS plotshown—FIG. 3E). A standard 10× field of view used in the tabulation ofCARMA phagocytosis function is shown, either mRFP alone (FIG. 3F) oroverlay (FIG. 3G). FACS based mRFP/GFP double positive events weredefined as phagocytic events, and were validated as such by AmnisImagestream FACS analysis. Events shown are gated on double positiveevents and ordered from high to low by the Amnis Imagestreamphagocytosis-erode algorithm (FIG. 3H). Phagocytosis of tumor cells bymRFP+CARMA in the THP-1 cell line model was further demonstrated byconfocal microscopy, verifying that GFP+ tumor cells have beencompletely enclosed within phagosomes via three-dimensional confocalz-stack reconstructions (FIGS. 31 and 3J). FIG. 3K demonstrates the fateof a single CARMA cell over time—with contact and immunological synapseformation being the first step, leading to phagocytic engulfment,degradation of tumor using loss of GFP as a marker of cell death,phagosome breakdown, and phagosome repair—demonstrating that CARMAsurvive post tumor cell phagocytosis. The data herein demonstrate thecapacity for CARMA to polyphagocytose many tumor cells at once.

Anti-CD19 CAR macrophages were tested using in an in vitro phagocytosisassay against CD19+(target) or CD19−(control) GFP+K562 tumor cells.Demonstrating the antigen specificity of CARMA, only antigen-bearingtumor cells were phagocytosed (FIG. 4A). To demonstrate the requirementfor the intracellular signaling domain in CARMA function, CAR19-Δζconstructs (which lack an intracellular signaling domain) were utilized.CAR19-Δζ macrophages failed to phagocytose tumor cells and hadsignificantly reduced anti-tumor function via an in vitro luciferasebased specific lysis assay (FIGS. 4B and 4C). In vitro CARMAphagocytosis assays were performed in the presence of R406 (Sykinhibitor), cytochalasin D (actin polymerization inhibitor), orblebbistatin (non-muscle myosin IIA inhibitor). R406, cytochalasin D,and blebbistatin independently abrogated the phagocytic function ofCARMA, indicating that CAR signaling in macrophages is Syk dependent andresults in actin polymerization and NMIIA mediated phagocytic function(FIGS. 4D-4F).

FIG. 5A is a flow cytometric graph showing expression of CD47 on targettumor cell lines relative to isotype control. K562 and K562-CD19+(K19)were used in these experiments, both of which are high CD47 expressingcell lines.

FIG. 5B is a graph showing that the addition of anti-CD47 monoclonalantibody selectively enhanced CAR but not Wt macrophage mediatedphagocytosis of target antigen bearing tumor cells. Wt or CAR19ζmacrophages were incubated with CD19+K562 tumor cells either with 0,0.01, 0.10, 1.00, or 10.0 mcg/mL anti-CD47 monoclonal antibody.

FIG. 5C is a graph showing that the addition of anti-SIRPα monoclonalantibody selectively enhanced CAR but not Wt macrophage mediatedphagocytosis of target antigen bearing tumor cells. Wt or CAR19ζmacrophages were incubated with CD19+K562 tumor cells either with 0,0.01, 0.10, 1.00, or 10.0 mcg/mL anti-SIRPα monoclonal antibody.

FIG. 5D is a graph demonstrating that blockade of the CD47/SIRPα axiswith anti-SIRPα monoclonal antibodies enhanced the polyphagocytic(defined as a macrophage that has engulfed 2 or more tumor cells atonce) by CAR macrophages.

To control for the added opsonization by the CD47/SIRPα blockingmonoclonal antibodies, a control anti-CD47 monoclonal antibody (clone2D3), which binds CD47 but does not block the CD47 to SIRPα bindingsite, was used in an in vitro phagocytosis assay. Only the clone whichblocked the binding site (Anti-CD47 clone B6H12) or blockade of theSIRPα receptor directly lead to enhancement of CARMA tumor phagocytosis(FIG. 5E).

To test whether blockade of the CD47/SIRPα axis on CAR macrophages leadsto loss of antigen specificity, an in vitro phagocytosis againstantigen-negative (CD19 negative) tumor cells was conducted in thepresence of Anti-CD47 or Anti-SIRPα monoclonal antibody, and there wasno observable phagocytosis (FIG. 5F).

The specificity of CARMA phagocytic enhancement in the presence of SIRPablocking monoclonal antibody was tested by knocking out the SIRPαreceptor on THP1 macrophages, and comparing tumor phagocytosis by CARMAor SIRPα-KO CARMA in the absence or presence of anti-SIRPα antibody.CRISPR/Cas9 was used for SIRPα deletion, and cells were sorted for SIRPαnegativity prior to functional assays. Knocking out SIRPα enhanced CARMAfunction, and adding anti-SIRPα back to the knockout cells failed tofurther enhance phagocytosis (FIG. 5G).

FIG. 6A demonstrates the specific lysis of CD19+ GFP+ Luciferase+K562cells by CAR19ζ CARMA but not Wt macrophages (using the THP-1 macrophagemodel) in an in vitro luciferase based killing assay at 48 hours in adose dependent manner.

FIG. 6B is a graph demonstrating the specific lysis of tumor cells byCAR19ζ or Wt THP-1 monocytes (undifferentiated, thus a model ofmonocytes rather than macrophages) in an in vitro luciferase basedkilling assay at 48 hours in a dose dependent manner.

FIG. 6C is a panel of images showing the luciferase drivenbioluminescence, derived from luciferase positive CD19+K562 tumor cells,after 48-hour co-culture with Wt or CAR19ζ macrophages in vitro, in theabsence or presence of 10 mcg/mL anti-SIRPα monoclonal antibody. FIG. 6Dis a graph demonstrating the specific lysis of Wt or CAR19ζ macrophages+/−anti-SIRPα monoclonal antibody.

CAR constructs with an FcεRI common γ (CAR19γ, CARMA19γ) subunitintracellular domain were generated, packaged into lentivirus, and usedto transduce THP-1 myeloid cells in a three-fold serial viral dilution.CAR19γ was expressed on THP-1 macrophages (FIG. 7A).

CAR19γ macrophages or CAR19ζ macrophages were sorted for 100% CARpositivity and utilized for in vitro functional characterization. CAR19ζand CAR19γ macrophages both phagocytosed CD19+ tumor cells, and bothdisplayed synergy with blockade of the CD47/SIRPα axis by the additionof anti-SIRPα monoclonal antibody (FIG. 7B).

CAR19ζ and CAR19γ macrophages both signal via Syk to drive tumorphagocytosis, as demonstrated in an R406 Syk inhibition in vitrophagocytosis assay (FIG. 7C).

Both CAR19ζ and CAR19γ THP1 macrophages, but not Wt THP1 macrophages,efficiently killed CD19+ tumor cells in an in vitro luciferase-basedspecific lysis assay after 24 hours of co-culture at various E:T ratios(FIG. 7D).

As white blood cells of the innate immune system, macrophages respond toconserved molecular cues of infection, such as pathogen associatedmolecular patterns, via constitutively expressed pathogen recognitionreceptors. Toll-like receptors are the best characterized pathogenrecognition receptors, and are known to activate macrophages.

To enhance the tumor phagocytic function of CARMA, in vitro phagocytosisassays were conducted using CAR macrophages that were primed with theligands for TLR1-9, independently, or media control. Ligands for TLR1,2, 4, 5, and 6 enhanced the phagocytic function of CARMA (FIG. 8A). Thissuggests that TLR ligands can be used to prime CARMA during theproduction process, or, TLR signaling domains can be encoded into theCAR construct to augment CAR signaling and downstream effector functionas a novel second/subsequent generation CARMA construct.

FIGS. 8B and 8C demonstrate that the difference between TLR ligands thatenhance or do not enhance CARMA phagocytosis of tumor cells holds trueat a wide range of TLR3 or TLR6 ligand concentrations.

β-glucan, a yeast product, binds to Dectin-1 on the surface ofmacrophages and results in activation and effector function. In order totest the capacity of β-glucan to augment CARMA function, in vitro tumorphagocytosis assays were conducted in the absence of presence of 5mcg/mL β-glucan. β-glucan enhanced the phagocytic capacity of CAR butnot Wt macrophages (FIG. 9A).

To test the capacity of β-glucan to enhance CARMA tumor killing, invitro luciferase based specific lysis assays were conducted at variousE:T ratios, in the presence of 0, 0.5, 5, of 50 mcg/mL β-glucan.β-glucan enhanced the specific lysis of antigen bearing tumor cells byCAR but not Wt THP-1 macrophages (FIG. 9B). These results indicate thatβ-glucan can be used as an adjuvant during the production process ofCARMA, or, the Dectin-1 intracellular signaling domain can be encodedinto the CAR transgene.

Given that β-glucan enhanced the function of CARMA, CAR constructscomprised of a Dectin-1 intracellular signaling domain were generated(FIG. 10A). These constructs were packaged into lentivirus and used totransduce THP-1 myeloid cells in a three-fold serial dilution oflentiviral titers. CAR was detected on the surface in both theCD8TM-Dectin1 CAR and DectinTM-Dectin1 CAR constructs (FIGS. 10B and10C). Cells were sorted for 100% positivity and utilized for downstreamin vitro functional experiments.

CD8TM-Dectin1 CAR and DectinTM-Dectin1 CAR macrophages were tested in anin vitro luciferase killing assay. Both constructs demonstrated specificlysis of tumor cells (10D). Dectin1-CAR macrophages were tested in an invitro tumor phagocytosis assay against K562 (control) or K19 (target)tumor cells, and Dectin1-CAR macrophages selectively phagocytosedcognate-antigen bearing tumor cells (FIG. 10E). Dectin-1 CAR macrophagesdemonstrated the capacity for phagocytosis of multiple tumor cells (FIG.10F).

In an in vitro tumor phagocytosis assay, Dectin1-CAR macrophagesdemonstrated synergy with blockade of SIRPα, or with priming with a TLRligand (FIG. 10G).

FIG. 11A is a graph showing calreticulin levels in three different CD19+target cell lines relative to isotype control. FIG. 11B is a graphshowing the normalized mean fluorescent intensity of calreticulinexpression in three different CD19+ target cell lines.

FIG. 11C is a graph showing that low levels of calreticulin moderatelyprotected target cells, specifically Nalm6 and JEKO cell lines, fromCAR19z macrophage phagocytosis. These data suggest that exploitation ofcalreticulin deposition/induction can be used an additional tactic toaugment CARMA effector function.

To validate and test the function of CAR in primary human monocytederived macrophages, several gene delivery approaches were tested. InFIG. 12A, anti-HER2 CAR constructs were cloned into mRNA expressionplasmids, transcribed in vitro, and the mRNA was directly electroporatedinto primary human monocytes. FIG. 13A demonstrates the gating strategy,viability, and 84.3% transfection efficiency relative to mockelectroporated cells. FIG. 12B demonstrates the efficiency of anti-HER2CAR mRNA electroporation into primary human monocyte derived macrophages(fully differentiated) at 79.7%.

FIG. 12C is a graph demonstrating that while mRNA electroporationresults in a high CAR transfection efficiency of both monocytes andmacrophages, CAR expression is temporary due to mRNA degradation,peaking at day 2 and disappearing by day 7 post electroporation invitro.

NSGS mice were injected with 1E6 SKOV3 CBG/GFP+ human ovarian cancercells via IP injection, a model of metastatic intraperitonealcarcinomatosis of HER2+ ovarian cancer. Mice were co-injected witheither mock electroporated or anti-HER2 CAR mRNA electroporated primaryhuman monocytes or primary human macrophages (1:1 E:T ratio) and tumorburden was imaged. CAR macrophages (FIG. 13A) and CAR monocytes (FIG.13B) demonstrated marginal reduction in tumor growth over approximatelytwo weeks. The first time point at which tumor burden wasbioluminescently quantified was 24 hours post-treatment, demonstratingthat CAR monocytes and macrophages had activity in the first 24 hours.

Lentiviral delivery of CAR transgenes to primary human monocyte derivedmacrophages was tested using multiple CAR constructs. In FIG. 14A, CAR19was delivered to human macrophages via lentiviral transduction,demonstrating a 4.27% and 38.9% transduction efficiency in the controlvs. CAR19 lentivirus (MOI 10) groups, respectively. The FACS gatingstrategy is shown.

FIG. 14B is a representative FACS plot showing the expression ofanti-HER2 CAR in primary human macrophages, with a 1.47 and 18.1%transduction efficiency between the control and MOI 10 CAR LVconditions, respectively.

Monocyte derived macrophages were generated by differentiating CD14+selected cells (from normal donor apheresis products) in GM-CSFconditioned media for 7 days. To optimize delivery of CAR via lentiviraltransduction, anti-CD19 and anti-HER2 lentiviruses were used totransduce macrophages at different points of the monocyte to macrophagedifferentiation process. Transduction efficiency peaked at the midpointof transduction (day 4), for both anti-CD19 and anti-HER2 CAR constructs(FIGS. 15A and 15B). Anti-CD19 CAR primary human macrophages were usedin an in vitro FACS based phagocytosis assay against CD19+ GFP+K562tumor cells, with CD11b+/GFP+ events being defined as phagocytic events.Macrophages transduced at different time points as in FIG. 15A were usedin this assay. FIG. 15C demonstrates that the efficacy of phagocytosistrended with the CAR transduction efficiency, peaking with macrophagestransduced at day 4 during the differentiation process.

Alternative transduction approaches to delivering transgenes to primaryhuman macrophages were tested, given that mRNA electroporation wastransient and lentivirus was only moderately efficient and required hightiter. Adenovirus (recombinant, replication deficient) was identified asan efficient approach to primary human macrophage transduction.Expression of Coxackie Adenovirus Receptor (the docking protein for Ad5)and CD46 (the docking protein for Ad35) were tested relative to isotypecontrol on primary human macrophages, and CD46 but not CoxackieAdenovirus receptor was highly expressed (FIG. 16A). Thus, chimericAd5f35 adenovirus was utilized for primary human macrophagetransduction, and was engineered via standard molecular biologytechniques to express a chimeric antigen receptor (GFP and empty Ad5f35viruses were used as controls) against HER2.

FIG. 16B shows that at an MOI of 1000, Ad5f35 effectively delivered atransgene (GFP was used as a model transgene) into human macrophages,and expression went up over time as monitored by GFP signalquantification on an IVIS Spectrum. FIG. 16C compares the transductionkinetics of primary human macrophages at different timepoints across abroad range of MOIs—up to 10,000.

FIG. 16C shows representative FACS plots of anti-HER2 CAR expression onAd5f35 transduced human macrophages 48 hours post transduction, at abroad range of viral MOIs.

FIG. 16D shows representative fluorescent microscopy images ofAd5f35-GFP transduced primary human macrophages, with the highesttransduction efficiency demonstrated at an MOI of 1000.

Primary human CARMA were tested in an in vitro phagocytosis assay viaFACS analysis. Macrophages (untransduced or anti-HER2 CAR) were stainedwith DiI prior to co-culture with GFP+ SKOV3 ovarian cancer cells.Phagocytosis, defined by DiI/GFP double positive events, was measured ata level of 26.6% in the CAR group and 4.55% in the control group (FIG.17A). To validate that the DiI/GFP double positive events werephagocytic events and not doublets, cytochalasin D (a phagocytosisinhibitor) was added to an arm of the experiment, and fully abrogatedCAR mediated phagocytosis down to 1.74%. To further validate thatprimary human CAR macrophages could phagocytose tumor cells, doublepositive events were gated by Amnis Imagestream FACS and ordered fromhigh to low by the Amnis phagocytosis-erode algorithm, demonstratingvisually that these double positive events represent phagocytosis (FIG.17B). In addition, DiI stained CAR-HER2 macrophages were co-culturedwith SKOV3-GFP and imaged by confocal microscope and phagocytosis wasverified.

Anti-HER2 CAR primary human macrophages were generated using Ad5f35-CARtransduction of monocyte derived macrophages. These cells (or controluntransduced cells) were utilized as effectors in an in vitro FACS basedphagocytosis assay of SKBR3 human breast cancer cells. FIG. 18demonstrates that CAR but not UTD human macrophages phagocytose breastcancer cells. In addition, addition of anti-SIRPα monoclonal antibodyenhanced CARMA but not UTD macrophage phagocytosis of breast cancercells. These results demonstrate that the synergy between blockade ofthe CD47/SIRPα axis seen with CARMA in the THP-1 model translates toprimary human macrophage studies.

Macrophages are white blood cells of the innate immune system and thushave sentinel anti-microbial properties. In order to demonstrate thatCAR macrophages are still functional innate immune cells in theanti-microbial sense, and do not lose the capacity to respond toinfectious stimuli, control untransduced or CAR macrophages wereemployed in a FACS based E. Coli phagocytosis assay. FIG. 19 is arepresentative FACS plot showing that CARMA exhibit intact phagocytosisof pH-Rodo Green E. Coli particles.

Primary human anti-HER2 CARMA were tested as effector cells in in vitroluciferase based killing assays. Anti-HER2 CARMA, but not control UTDmacrophages, led to the specific lysis of HER2+K562 cells but notcontrol K562 cells, lacking HER2 expression, after 48 hours ofco-culture (FIG. 20A). To demonstrate that CARMA killing can betranslated to tumor cells expressing HER2 at physiologic levels (asopposed to K562-HER2 which is lentivirally transduced to overexpressHER2), SKBR3 breast cancer cells and SKOV3 ovarian cancer cells wereused as targets. CARMA, but not control UTD or control Empty Ad5f35transduced macrophages, had significant anti-tumor activity against bothmodels after 48 hours of co-culture (FIGS. 20B and 20C). In order totest synergy between blockade of the CD47/SIRPα axis in a killing assay,SKOV3 ovarian cancer cells were co-cultured with media, controluntransduced macrophages, anti-HER2 CARMA, anti-HER2 CARMA+antiCD47 mAB(10 mcg/mL), or anti-HER2 CARMA+anti-SIRPα (10 mcg/mL) and luciferasesignal was serially measured. CARMA led to complete tumor eradication byday 13, while the kinetics of tumor eradication were even faster in thepresence of blocking the CD47/SIRPα axis (FIG. 20D). Synergy withβ-glucan, which was demonstrated in a THP-1 macrophage CARMA model, wastested in a similar experiment, and β-glucan priming of the CARMA led toenhanced tumor killing kinetics (FIG. 20E). Exposure of CARMA to LPS (aTLR-4 ligand) or Poly-IC (a TLR-3 ligand) led to modulation of theanti-tumor effect (FIG. 20F).

The capacity for primary human CARMA to clear tumors in vitro wasdemonstrated by luciferase assay in FIGS. 20A-20F. To validate theseresults, GFP+ SKOV3 ovarian cancer cells were co-cultured with controlUTD macrophages, control UTD macrophages plus 10 mcg/mL trastuzumab,control empty Ad5f35 virus transduced macrophages, or anti-HER2 primaryhuman CARMA. CARMA, but not the control conditions, were capable ofclearing the tumor cells (FIG. 21).

Macrophages are phenotypically plastic cells capable of adopting diversefunctional features, commonly separated into the M1 and M2 macrophageclassifications—with M1 being inflammatory/activated, and M2 beingimmunosuppressive/tumor-promoting. 48 hours after transduction ofprimary human macrophages with Ad5f35 CAR virus, a dose dependentup-regulation of M1 markers CD80/CD86, and a dose dependentdown-regulation of M2 marker CD163, were measured by FACS (FIG. 22A). Totest whether this effect was a result of CAR expression or Ad5f35transduction, macrophages were transduced with either nothing, emptyAd5f35, or anti-HER2 Ad5f35, and empty/CAR Ad5f35 showed the samepattern of phenotype shift (FIG. 22B).

The solid tumor microenvironment is generally immunosuppressive and canlead to macrophage polarization to the M2 state. To test whether CARMA,which is M1 polarized due to viral transduction, is resistant toimmunosuppressive cytokine mediated subversion to M2, controluntransduced or anti-HER2 CAR human macrophages were exposed to IL-4,IL-10, or IL-13 for 24 hours prior to co-culture with SKOV3 ovariancancer cells. Control UTD macrophages conditioned with suppressivecytokines led to the enhancement of tumor growth, while CARMA exposed tosuppressive cytokines maintained their killing activity in a luciferasebased in vitro specific lysis assay at 48 hours (FIG. 22C).

To further test the resistance to immunosuppression of human CARmacrophages, control UTD, Empty Ad5f35, or anti-HER2 CAR Ad5f35transduced macrophages were exposed to 10 ng/mL of IL-4, a canonical M2inducing cytokine, or cancer cells that were previously shown to subvertmacrophages to M2 during co-culture (SKOV3, ovarian cancer cell line;HDLM2, Hodgkin lymphoma cell line). Control UTD macrophages upregulatedCD206, an M2 marker that specifically responds to IL-4 stimulation viaSTATE phosphorylation. Empty Ad5f35, and more so CAR-Ad5f35 transducedmacrophages, displayed resistance to IL-4 and tumor induced subversionto the M2 phenotype (FIG. 22D).

In order to further characterize the phenotype of CAR macrophages, themetabolic phenotype was probed using the Seahorse assay to measureoxygen consumption. M2 macrophages have a higher basal oxygenconsumption rate than M0 or M1 macrophages, due to a higher reliance onoxidative phosphorylation for ATP production. Control UTD or anti-HER2CAR macrophages were exposed to IL-4 for 24 hours to polarize to M2 (ornot), and oxygen consumption rate was measured. Control UTD macrophagesdemonstrated the characteristic increased basal oxygen consumptioncharacteristic of M2 macrophages, while CARMA did not respond to IL-4,suggesting that it is resistant to M2 subversion (FIG. 22E). These datacombined illustrate, using phenotypic, metabolic, and functional assays,that CARMA are resistant to M2 subversion.

Primary human normal donor monocytes (purified via CD14 positiveselection) were transduced with Ad5f35-CAR-HER2 at MOI's ranging from 0(UTD) to 1000. CAR expression was measured via FACS 48 hours posttransduction. CAR monocytes were efficiently generated with Ad5f35, withexpression peaking at an MOI of 1000 (FIGS. 23A and 23B). Monocytesmaintained high viability (measured by FACS Live/Dead Aqua analysis) atMOIs up to 1000 (FIG. 23C). CAR but not untransduced (UTD) humanmonocytes upregulated M1 activation markers (FIG. 23D) and downregulatedM2 markers (FIG. 23E), as analyzed by FACS, demonstrating an M1 monocytephenotype 48 hours post transduction.

Anti-HER2 CAR monocyte killing was assessed via in vitro luciferasebased killing assay at a range of effector:target (E:T) ratios.Untransduced (UTD) or CAR-HER2-zeta (CAR) monocytes were co-culturedwith either HER2+ SKBR3 (human breast cancer) or HER2+ SKOV3 (humanovarian cancer) cells in vitro. Specific lysis was calculated anddetermined at 24, 48, and 96 hours post initiation of co-culture. CARbut not UTD monocytes lysed both breast and ovarian cancer cells invitro (FIGS. 24A and 24B).

NOD-scid IL2Rg-null-IL3/GM/SF, NSG-SGM3 (NSGS) mice were used to modelhuman HER2(+) ovarian cancer xenografts in vivo. On day 0 mice wereinjected intraperitoneally (IP) with 7.5E5 click beetle green luciferase(CBG luc) positive/green fluorescent protein (GFP) positive SKOV3ovarian cancer cells as a model of intraperitoneal carcinomatosis, anaggressive inherently metastatic model of solid malignancy. Mice wereeither untreated (tumor alone), or injected with a single dose of 4E6untransduced (UTD) or CAR-HER2 (CARMA) human macrophages on day 0 via IPinjection (schematic diagram, FIG. 25A). Mice were serially imaged usingbioluminescence (total flux; photons/second) as a surrogate of tumorburden. Mice that received CARMA treatment had a decrease in tumorburden of approximately two orders of magnitude (FIGS. 25B and 25C).Mice treated with CARMA had a 30 day survival benefit (p=0.018) relativeto untreated or UTD macrophage treated mice (FIG. 25D). To demonstratetrafficking of macrophages into the solid tumor nodule, tumors wereharvested from mice that died on day 36 and assessed for the presence ofadoptively transferred human macrophages via human CD45 expression onFACS analysis (FIG. 25E).

Human macrophages were either untransduced (UTD) or transduced withempty Ad5f35 virions lacking a transgene (Empty) or Ad5f35-CAR-HER2-ζ(CARMA) at multiplicities of infection of 1000. Surface CAR expressionwas verified by FACS analysis 48 hours post transduction (FIG. 26A).Surface markers were assessed to demonstrate M1 macrophage polarizationin cells transduced by either empty Ad5f35 or CAR-HER2-ζ Ad5f35. M1markers (HLA DR, CD86, CD80, PDL1) were upregulated while M2 markers(CD206, CD163) were downregulated (FIG. 26B). NSGS mice were again usedin an IP model of HER2+ metastatic ovarian cancer, and were stratifiedinto four treatment arms (n=5 per arm). Mice were left untreated orgiven IP injections of 1E7 untransduced, empty-Ad5f35 transducedmacrophages, or CAR-HER2-ζ transduced macrophages on day 0 (FIG. 26C).Tumor burden was monitored via serial bioluminescent imaging, withrepresentative data shown at day 27 post tumor engraftment (FIGS. 26Dand 26E). CARMA treated mice had roughly 2,400 fold less tumor burdenthan untreated mice at day 20 post treatment.

NSGS mice were used in an IP model of HER2+ metastatic ovarian cancer,and were stratified into four treatment arms (n=5 per arm), including notreatment, and either 3E6, 1E7, or 2E7 CAR-HER2-ζ human macrophages,administered IP on day 0 (FIG. 27A). Tumor burden was monitored viaserial bioluminescent imaging, and a macrophage number dependent doseresponse was observed in this model (FIG. 27B). Single doses of CAR-HER2macrophages at 3E6, 1E7, or 2E7 macrophages per mouse led to dosedependent tumor eradication (relative to untreated mice) by day 36 postengraftment (FIG. 27C).

FIG. 28 is an illustration of the proposed therapeutic approach forCARMA. In brief, patient monocytes would be selected from the peripheralblood, ex vivo differentiated and transduced to express a CAR,co-stimulated (or not) with synergistic compounds, and injected backinto the patient either intravenously, intraperitoneally,intratumorally, via interventional radiological procedure, or by otherroute. Of note, the differentiated process could be skipped andmonocytes can be transduced and infused back into the patient. Themonocyte source may also be an HLA matched donor.

OTHER EMBODIMENTS

The recitation of a listing of elements in any definition of a variableherein includes definitions of that variable as any single element orcombination (or subcombination) of listed elements. The recitation of anembodiment herein includes that embodiment as any single embodiment orin combination with any other embodiments or portions thereof.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety. While this invention has been disclosed with referenceto specific embodiments, it is apparent that other embodiments andvariations of this invention may be devised by others skilled in the artwithout departing from the true spirit and scope of the invention. Theappended claims are intended to be construed to include all suchembodiments and equivalent variations.

1-44. (canceled)
 45. A modified cell comprising a chimeric antigenreceptor (CAR), wherein the CAR comprises an antigen binding domain, atransmembrane domain and an intracellular domain of a stimulatory and/orco-stimulatory molecule, and wherein the modified cell expresses the CARand possesses targeted effector activity, wherein the modified cellcomprises a macrophage or a monocyte, and wherein the modified cellcomprises a lentiviral component.
 46. The modified cell of claim 45,wherein the modified cell exhibits reduced SIRPα activity relative to anunmodified monocyte or macrophage.
 47. The modified cell of claim 45,wherein the antigen binding domain of the CAR comprises an antibodyselected from the group consisting of a monoclonal antibody, apolyclonal antibody, a synthetic antibody, human antibody, humanizedantibody, single domain antibody, single chain variable fragment, andantigen-binding fragments thereof.
 48. The modified cell of claim 45,wherein the antigen binding domain of the CAR is selected from the groupconsisting of an anti-CD19 antibody, an anti-HER2 antibody, ananti-mesothelin antibody, an anti-PSMA antibody, and a fragment thereof.49. The modified cell of claim 45, wherein the transmembrane domain ofthe CAR comprises a CD8 or CD28 transmembrane domain.
 50. The modifiedcell of claim 45, wherein the intracellular domain of the CAR comprisesdual signaling domains.
 51. The modified cell of claim 45, wherein theintracellular domain of the CAR comprises a CD3 zeta intracellulardomain.
 52. The modified cell of claim 45, wherein the targeted effectoractivity is directed against a target cell comprising an antigen thatspecifically binds the antigen binding domain of the CAR.
 53. Themodified cell of claim 45, wherein the targeted effector activity isselected from the group consisting of phagocytosis, targeted cellularcytotoxicity, antigen presentation, and cytokine secretion.
 54. Themodified cell of claim 45, further comprising an agent selected from thegroup consisting of a nucleic acid, an antibiotic, an anti-inflammatoryagent, an antibody or antibody fragments thereof, a growth factor, acytokine, an enzyme, a protein, a peptide, a fusion protein, a syntheticmolecule, an organic molecule, a carbohydrate or the like, a lipid, ahormone, a microsome, a derivative or a variation thereof, and anycombination thereof.
 55. The modified cell of claim 45, wherein themodified cell has at least one upregulated M1 marker and at least onedownregulated M2 marker.
 56. The modified cell of claim 45, wherein themodified cell is genetically modified to express the CAR.
 57. Themodified cell of claim 45, wherein the targeted effector activity isenhanced by inhibition of CD47 or SIRPα activity.
 58. A pharmaceuticalcomposition comprising the cell of claim 45 and a pharmaceuticallyacceptable carrier.
 59. The pharmaceutical composition of claim 58,wherein at least 35% of the cells in the composition express a CAR. 60.A method for stimulating an immune response to a target tumor cell ortumor tissue in a subject comprising administering to a subject atherapeutically effective amount of a pharmaceutical compositioncomprising the modified cell of claim
 45. 61. A method of modifying acell, comprising: introducing a chimeric antigen receptor (CAR) into thecell, wherein the CAR comprises an antigen binding domain, atransmembrane domain and an intracellular domain of a stimulatory and/orco-stimulatory molecule, and wherein the modified cell expresses the CARand possesses targeted effector activity, wherein the modified cellcomprises a macrophage or a monocyte, and wherein the modified cellcomprises a lentiviral component.
 62. The method of claim 61, whereinintroducing the CAR into the cell comprises introducing a nucleic acidsequence encoding the CAR.
 63. The method of claim 61, whereinintroducing the nucleic acid sequence comprises electroporating an mRNAencoding the CAR.
 64. The method of claim 61, wherein introducing thenucleic acid sequence comprises transducing the cell with a viral vectorcomprising a nucleic acid sequence encoding the CAR.
 65. The method ofclaim 61, wherein the targeted effector activity is selected from thegroup consisting of phagocytosis, targeted cellular cytotoxicity,antigen presentation, and cytokine secretion.
 66. The method of claim61, further comprising inhibiting CD47 or SIRPα activity to enhance thetargeted effector activity.
 67. The method of claim 66, whereininhibiting CD47 or SIRPα activity comprises contacting the cell with ablocking anti-CD47 antibody or a blocking anti-SIRPα antibody.
 68. Themethod of claim 61, wherein the intracellular domain of the CARcomprises dual signaling domains.
 69. The method of claim 61, whereinthe intracellular domain of the CAR comprises a CD3 zeta intracellulardomain.
 70. The method of claim 61, wherein the antigen binding domainof the CAR comprises an antibody selected from the group consisting of asynthetic antibody, human antibody, humanized antibody, single domainantibody, single chain variable fragment, and antigen-binding fragmentsthereof.
 71. The method of claim 61, wherein the antigen binding domainof the CAR is selected from the group consisting of an anti-CD19antibody, an anti-HER2 antibody, an anti-mesothelin antibody, ananti-PSMA antibody, and a fragment thereof.